Clinical application of amplicon-based next-generation sequencing to therapeutic decision making in lung cancer.

BACKGROUND: The clinical implementation of genomic profiling for lung cancer with high-throughput, multiplex tests is warranted to allow prioritization of appropriate therapies for individual patients. We have now applied such testing to detect actionable mutations that may inform treatment recommendations in lung cancer. PATIENTS AND METHODS: We prospectively applied amplicon sequencing panels that cover both mutational hotspots in 22 genes related to lung and colon tumorigenesis as well as 72 major variants of ALK, RET, ROS1, and NTRK1 fusion transcripts. We then determined the proportion of patients who received genotype-directed therapy and their overall survival (OS). RESULTS: Tumor specimens from 110 patients with lung cancer recruited between July 2013 and March 2015 were analyzed. The most common genetic alterations were TP53 mutations in 42 patients, followed by EGFR mutations in 25, STK11 mutations in 12, and KRAS mutations in 10. Potentially actionable mutations were identified in 44 patients including 50% of those with adenocarcinoma and 14% of those with squamous cell carcinoma. The OS of patients with advanced or recurrent cancer who had an actionable mutation and received targeted therapy (median OS not achieved) was significantly longer than that of those with no mutation (18.1 months, P = 0.041) or of those with a mutation not so treated (6.1 months, P = 0.0027). CONCLUSIONS: Multiplex genomic testing was performed on formalin-fixed, paraffin-embedded tumor specimens with a success rate of ≥95%. Such testing can assist physicians in matching patients with approved or experimental targeted treatments. CLINICAL TRIAL REGISTRATION: The University Medical Hospital Information Network (UMIN) Clinical Trials Registry under the identifier UMIN000014782.

A phase I dose-escalation study of eribulin and S-1 for metastatic breast cancer.

BACKGROUND: We evaluated the safety, maximum-tolerated dose (MTD), pharmacokinetics, recommended dose for phase II (P2RD), and preliminary anticancer activity of a combination eribulin and S-1 therapeutic in metastatic breast cancer patients pretreated with anthracycline and taxane. METHOD: Patients aged 20-74 years were recruited. In level 1, patients received S-1 (65 mg m(-2)) from day 1 to 14, and eribulin (1.1 mg m(-2)) on day 1 and 8 in a 21-day cycle. In level 2, eribulin was increased to 1.4 mg m(-2). In level 3, S-1 was increased to 80 mg m(-2). RESULTS: Twelve patients were enrolled into three cohorts. Planned dose escalation was completed, with one case exhibiting dose-limiting toxicity (grade 3 hypokalaemia) at level 3, without reaching the MTD. The P2RD was determined to be level 2 (eribulin 1.4 mg m(-2) and S-1 65 mg m(-2)). The most common grade 3 or 4 toxicity was neutropenia (83.3%), followed by febrile neutropenia (25.0%). Five of eleven patients (41.7%) with measurable disease had a partial response. Pharmacokinetics were characterised by dose-dependent elimination and nonlinear exposure. CONCLUSION: Dose level 3 was not tolerated owing to febrile neutropenia development. Thus, intermediate dose level 2 was recommended for further evaluation. Preliminary antitumour activity warrants further investigation in this setting.

Site-specific changes in cytokine response to septic dose of lipopolysaccharide in ovariectomized female rats.

PURPOSE: The immune response is altered according to hormonal and metabolic status. Obesity increases the inflammatory and fever response, whereas loss of gonadal steroid decreases behavioral response to immune stress. However, the immune systems of ovariectomized animals exhibiting obesity and gonadal steroid deficiency, particularly under septic conditions, have not been fully examined. In the present study, we evaluated the ovariectomy-induced changes of central and peripheral immune responses to life-threatening septic stimulus. METHODS AND RESULTS: Ovariectomized rats showed heavier body weight and lighter uterine weight when compared with gonadally intact rats. Fever response to septic dose of lipopolysaccharide (LPS) in ovariectomized rats was less evident when compared with that in gonadally intact rats. In addition, under LPS-injected septic conditions, hypothalamic gene levels of Interleukin-1ß (IL-1ß), Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) and serum protein levels of IL-1ß and TNF-α in ovariectomized rats were lower than those in gonadally intact rats. On the other hand, IL-6 levels in visceral fat under septic conditions were higher in ovariectomized rats than in gonadally intact rats. CONCLUSIONS: These findings indicate that ovariectomy-induced site-specific changes in cytokine response under septic conditions. As hypothalamic, but not peripheral, pro-inflammatory cytokines are directly involved in the fever response, the attenuation of fever response observed in ovariectomized rats may be caused by a reduction in central cytokine responses.

Ovarian torsion associated with cessation of hormonal treatment for polycystic ovarian syndrome: a case report.

Torsion of an ovary or fallopian tube (adnexal torsion) usually occurs in ovaries with tumors or functional cysts. In polycystic ovarian syndrome (PCOS), the ovaries are bilaterally enlarged, but these enlarged ovaries rarely twist. Recently, the authors encountered a PCOS patient with ovarian torsion after the cessation of Kaufmann treatment. The etiological factors were unclear, but the authors suggest that the increase in ovarian volume was due to transient hypergonadotropic feedback. Thus, more attention should be paid to adnexal torsion that may arise subsequent to transient hypergonadtropic states, in relation to the cessation of hormonal treatment, and enlarged ovaries in PCOS patients.

Amniocentesis can be useful during the third trimester of pregnancy for antenatal diagnosis of Pallister-Killian syndrome: a case report.

Pallister-Killian syndrome (PKS) is an extremely rare genetic disease characterized cytogenetically by tetrasomy 12p mosaicism. We recently encountered a case of maternal hydramnios associated with congenital diaphragm hernia according to the prenatal diagnosis. Prenatal diagnosis revealed a non-mosaic 47, XY, i(12)(p10) karyotype at amniocentesis of G-band and M-FISH analysis. We performed chromosomal analysis in both interphase and metaphase cells from a cord blood lymphocyte specimen. Mosaic tetrasomy of chromosome 12p was supported by G-banding or FISH analysis. When fetal observations are performed in detail using 2D/3D US, PKS may be diagnosed. In addition, it is effective to perform amniocentesis during the third trimester of pregnancy.

Multiple ulcers in the small and large intestines occurred during tocilizumab therapy for rheumatoid arthritis.

Tocilizumab is a monoclonal antibody against human interleukin-6 receptor which blocks the binding of interleukin-6 to its receptor. Tocilizumab is effective for the treatment of inflammatory disorders including rheumatoid arthritis. We report a case of multiple ulcers in the small and large intestines, which occurred during tocilizumab therapy. A 57-year-old woman started to use tocilizumab for rheumatoid arthritis. Three months later, she complained of hematochezia. Double-balloon endoscopy revealed multiple small aphthoid ulcers in the small and large intestines. One month after the woman had recovered, she was given tocilizumab again. The woman had hematochezia and abdominal pain again 2 weeks later. Colonoscopy revealed multiple round, discrete punched-out ulcers in the terminal ileum, and vast deep ulcers from the cecum to the descending colon. Bioptic histopathology and cultivation showed non-specific findings. Six weeks after discontinuation of tocilizumab, ulcers in the small and large intestine dramatically improved, leaving ulcer scars. This disease course and the results of examination made us strongly suspect that tocilizumab induced multiple ulcers in the small and large intestines. Interleukin-6 is a pleiotropic cytokine and involved in intestinal mucosal wound healing as well as in inflammatory processes. It is possible that tocilizumab inhibited tissue repair of the intestine and caused intestinal ulcers.

Marked anti-tumour activity of the combination of YM155, a novel survivin suppressant, and platinum-based drugs.

BACKGROUND: Survivin, a member of the inhibitor of apoptosis protein family, is an attractive target for cancer therapy. We have now investigated the effects of the combination of YM155, a novel small-molecule inhibitor of survivin expression, and platinum compounds (cisplatin and carboplatin) on human non-small cell lung cancer (NSCLC) cell lines. METHODS: The anti-cancer efficacy of YM155 in combination with platinum compounds was evaluated on the basis of cell death and progression of tumour xenografts. Platinum compound-induced DNA damage was evaluated by immunofluorescence analysis of histone gamma-H2AX. RESULTS: Immunofluorescence analysis of histone gamma-H2AX showed that YM155 delayed the repair of double-strand breaks induced in nuclear DNA by platinum compounds. The combination of YM155 and platinum compounds also induced synergistic increases both in the number of apoptotic cells and in the activity of caspase-3. Finally, combination therapy with YM155 and platinum compounds delayed the growth of NSCLC tumour xenografts in nude mice to an extent greater than that apparent with either treatment modality alone. CONCLUSION: These results suggest that YM155 sensitises tumour cells to platinum compounds both in vitro and in vivo, and that this effect is likely attributable to the inhibition of DNA repair and consequent enhancement of apoptosis.

Effects of intrauterine undernutrition on hypothalamic Kiss1 expression and the timing of puberty in female rats.

Recent studies have suggested that intrauterine undernutrition is closely associated with the pathogenesis of diseases after birth. Perinatal undernutrition is known to disturb the development of reproductive function and delay the onset of puberty in some species. Using a rat model, we determined the effects of prenatal undernutrition on the development of the hypothalamic kisspeptin system and evaluated whether the alteration of the kisspeptin system contributes to the delayed onset of puberty induced by prenatal undernutrition. We also evaluated the effects of prenatal undernutrition on the developmental changes in serum leptin levels because leptin was a putative positive regulator of the hypothalamic kisspeptin system. We compared the timing of vaginal opening (VO) and the developmental changes in body weight, hypothalamic Kiss1 mRNA levels, and serum leptin concentrations between offspring with prenatal undernutrition (UN offspring) and normal nutrition (NN offspring). After birth, the UN offspring showed rapid growth and had caught up to body weight of the NN offspring by postnatal day 12. After postnatal day 16, the UN offspring showed significantly lower Kiss1 mRNA levels than the NN offspring, despite their significantly higher serum leptin levels (at days 20 and 28). The timing of VO in the UN offspring was delayed compared with that in the NN offspring, and chronic central injection of kisspeptin normalized the timing of VO in the UN offspring. These results suggest that decreased hypothalamic kisspeptin action contributes to the delayed onset of puberty in prenatally undernourished female rats. Increased leptin resistance in the kisspeptin system might be involved in these alterations.

Decreased expression of kisspeptin mediates acute immune/inflammatory stress-induced suppression of gonadotropin secretion in female rat.

Kisspeptin and its corresponding receptor, the G protein-coupled receptor 54, play an important role in reproductive systems. It has been suggested that reproductive disorders in metabolically disrupted animals are caused by the alteration of hypothalamic KiSS-1 systems. Immune/inflammatory challenge is also known to disrupt reproductive function. However, the effects of immune/inflammatory challenge on KiSS-1 systems have not been investigated. In this study, we showed that lipopolysaccharide (LPS) injection decreased hypothalamic KiSS-1 mRNA expression as well as plasma LH levels in ovariectomized rats. Indomethacin completely blocked the suppressive effects of LPS on LH secretion and KiSS-1 mRNA level. Furthermore, we showed that i.v. injection of kisspeptin increased plasma LH levels in LPS-administrated rats to the same degree as in saline-injected rats. These results suggest that KiSS-1 systems are sensitive to immune/inflammatory challenge conditions and transmit these signals into the central reproductive system.

A case of maxillary duplication with a soft palate reconstruction using a forearm flap.

Maxillary duplication is a rare congenital anomaly that occurs in the jaw/mouth area. It is generally regarded as sporadic in nature. Total or subtotal soft palate reconstruction for oropharyngeal defects, which include post-surgical and congenital defects, presents a difficult surgical challenge. A maxillary duplication in which the soft palate is reconstructed using a vascularized forearm flap is described. The velopharyngeal insufficiency in the present case is caused by the almost complete deficiency of the soft palate, suggesting that a conventional pharyngeal flap operation with localized mucosal myocutaneous flaps would not produce favorable results in terms of postoperative contractions in the pharyngeal flaps. In such cases, the reconstruction of the soft palate using vascularized free forearm flaps, guided by flexibility regarding the size and adequate thickness of the flaps, may be useful.

Enhancement of the antitumor activity of ionising radiation by nimotuzumab, a humanised monoclonal antibody to the epidermal growth factor receptor, in non-small cell lung cancer cell lines of differing epidermal growth factor receptor status.

The expression and activity of the epidermal growth factor receptor (EGFR) are determinants of radiosensitivity in several tumour types, including non-small cell lung cancer (NSCLC). However, little is known of whether genetic alterations of EGFR in NSCLC cells affect the therapeutic response to monoclonal antibodies (mAbs) to EGFR in combination with radiation. We examined the effects of nimotuzumab, a humanised mAb to EGFR, in combination with ionising radiation on human NSCLC cell lines of differing EGFR status. Flow cytometry revealed that H292 and Ma-1 cells expressed high and moderate levels of EGFR on the cell surface, respectively, whereas H460, H1299, and H1975 cells showed a low level of surface EGFR expression. Immunoblot analysis revealed that EGFR phosphorylation was inhibited by nimotuzumab in H292 and Ma-1 cells but not in H460, H1299, or H1975 cells. Nimotuzumab augmented the cytotoxic effect of radiation in H292 and Ma-1 cells in a clonogenic assay in vitro, with a dose enhancement factor of 1.5 and 1.3, respectively. It also enhanced the antitumor effect of radiation on H292 and Ma-1 cell xenografts in nude mice, with an enhancement factor of 1.3 and 4.0, respectively. Nimotuzumab did not affect the radioresponse of H460 cells in vitro or in vivo. Nimotuzumab enhanced the antitumor efficacy of radiation in certain human NSCLC cell lines in vitro and in vivo. This effect may be related to the level of EGFR expression on the cell surface rather than to EGFR mutation.

The type 2 corticotrophin-releasing hormone receptor mediates orexin A-induced luteinising hormone suppression in ovariectomised rats.

Orexins are thought to be regulatory factors of the arousal and sleep patterns. They also affect immune, feeding, autonomic and neuroendocrine systems. We have previously shown that intracerebroventricular (i.c.v.) injection of orexin decreases pulsatile luteinising hormone (LH) secretion in ovariectomised (OVX) rats. However, the details of this mechanism have not been fully examined. Intracerebroventricular injection of orexin A also stimulates corticotrophin-releasing hormone (CRH) systems, which have been implicated in the stress-induced suppression of reproductive function. In the present study, we investigated the role of CRH systems in orexin-induced LH suppression. OVX rats were implanted with i.c.v. and intravenous (i.v.) cannulae. After i.c.v. injection of orexin and/or CRH receptor antagonists, blood samples were collected through the i.v. cannula at 6-min intervals for 120 min for LH measurement. Intracerebroventricular injection of orexin A or B (3 nmol/2.5 microl) suppressed pulsatile LH secretion. Coadministration of orexin A and alpha-helical corticotrophic-releasing factor (CRF), a nonselective CRH receptor antagonist (13 nmol/2.5 microl), or astressin(2)B, a selective type2 (CRH-R2) CRH receptor antagonist (28 nmol/2.5 microl), partly restored pulsatile LH secretion. Orexin B-induced LH suppression was not restored by alpha-helical CRF. In addition, i.c.v. injection of orexin A increased CRH and urocortin II (UcnII), but not Ucn mRNA levels, in the hypothalamus. These findings suggest that CRH-R2 mediates orexin A-induced LH suppression and it is possible that CRH and UcnII in the hypothalamus are involved in this pathway.

The novel microtubule-interfering agent TZT-1027 enhances the anticancer effect of radiation in vitro and in vivo.

TZT-1027 is a novel anticancer agent that inhibits microtubule polymerisation and manifests potent antitumour activity in preclinical models. We have examined the effect of TZT-1027 on cell cycle progression as well as the anticancer activity of this drug both in vitro and in vivo. With the use of tsFT210 cells, which express a temperature-sensitive mutant of Cdc2, we found that TZT-1027 arrests cell cycle progression in mitosis, the phase of the cell cycle most sensitive to radiation. A clonogenic assay indeed revealed that TZT-1027 increased the sensitivity of H460 cells to gamma-radiation, with a dose enhancement factor of 1.2. Furthermore, TZT-1027 increased the radiosensitivity of H460 and A549 cells in nude mice, as revealed by a marked delay in tumour growth and an enhancement factor of 3.0 and 2.2, respectively. TZT-1027 also potentiated the induction of apoptosis in H460 cells by radiation both in vitro and in vivo. Histological evaluation of H460 tumours revealed that TZT-1027 induced morphological damage to the vascular endothelium followed by extensive central tumour necrosis. Our results thus suggest that TZT-1027 enhances the antitumour effect of ionising radiation, and that this action is attributable in part to potentiation of apoptosis induction and to an antivascular effect. Combined treatment with TZT-1027 and radiation therefore warrants investigation in clinical trials as a potential anticancer strategy.

[Successful resection of left ventricular myxoma in a toddler; report of a case].

A 4-year-old girl was found to have large left ventricular myxoma without any tumor-related symptoms. She underwent an urgent surgery and the myxoma was successfully removed through a left ventriculectomy. Great care was taken to prevent tumor-embolization during surgery, and to resect the endocardium attaching directly to the tumor. Future surveillance of this case warrants our operative technique described in this report.

An in vitro interethnic comparison of monoamine oxidase activities between Japanese and Caucasian livers using rizatriptan, a serotonin receptor 1B/1D agonist, as a model drug.

AIMS: Monoamine oxidase (MAO) is located in human liver, and catalyses the oxidative deamination step of many xenobiotics. However, whether there exists an interethnic difference in MAO activities has, to our knowledge, not been clarified. We aimed to assess the MAO type A (MAO-A) involvement in the metabolic pathway of rizatriptan (RIZ), an antimigraine 5-hydroxytryptamine (5-HT)1B/1D agonist, and the interethnic difference in MAO activities between Caucasians and Japanese using RIZ as a model drug in in vitro experiments. METHODS: Oxidative deaminase activities were determined with the subcellular fractions of Japanese livers and the microsomal fraction of Caucasian livers using RIZ, 5-HT (MAO-A substrate) and 2-phenylethylamine (PEA) (MAO-B substrate) as substrates. RESULTS: The oxidative deaminase activities of RIZ vs. 5-HT were highly (r = 0.87 and 0.96, P < 0.001) correlated with each other in both the microsomal and mitochondrial fractions of Japanese livers. Subsequent results were obtained from in vitro experiments using liver microsomes based upon these findings. The oxidative deaminase activities of RIZ were inhibited completely by the nanomolar-order concentration of clorgyline and Ro 41-1049 (MAO-A selective inhibitors), but not by that of Ro 16-6491 (MAO-B selective inhibitor). The majority of the mean Michaelis-Menten values for three substrates toward MAO obtained from six Japanese and six Caucasian liver microsomes reached no significant differences between the two ethnic groups. The mean microsomal oxidative deaminase activities assessed in 18 Japanese and 20 Caucasian livers using the three substrates also showed no significant differences between the two ethnic groups. CONCLUSIONS: RIZ is mainly metabolized by MAO-A and the in vitro oxidative deaminase activities mediated via MAO-A and -B do not appear to differ between Japanese and Caucasians.

Molecular cloning of Bombyx cerebral opsin (Boceropsin) and cellular localization of its expression in the silkworm brain.

We have cloned a cDNA for a novel opsin from the larval brain of the silkworm Bombyx mori in which the photoperiodic photoreceptor had been supposed to reside in the cephalic central nervous system (CNS). Its deduced amino acid sequence was composed of 381 amino acids and included amino acid residues highly conserved in insect visual pigments. This opsin belonged to the long wavelength photoreceptor group of insect opsins and showed the greatest degree of homology (84%) with the green visual photoreceptor in the sphingid moth. We have designated this Bombyx cerebral opsin as Boceropsin. Southern blotting experiments indicated that the Boceropsin gene is present in a single copy, and RT-PCR analysis revealed that Boceropsin mRNA is expressed in the larval brain but not in the subesophageal ganglion (Sg) or thoracic ganglion (Tg). Immunohistochemical analyses demonstrated that Boceropsin protein is present bilaterally in some defined cells localized in the brain of Bombyx larvae. This is the first report of expression of an opsin-based protein in CNS of an insect. The possibility that the Boceropsin functions as the photoperiodic receptive pigment in the silkworm is also discussed.

Formation of PCDDs and PCDFs during the combustion of polyvinylidene chloride.

In laboratory-scale combustion of polyvinylidene chloride (PVDC) with a quartz tubular furnace designed and fabricated to provide the desired combustion temperature and mixing state of combustion gas with air, it was found that at 800 degrees C or higher the level of polychlorinated dibenzo-p-dioxins/polychlorinated dibenzofurans [corrected] (PCDDs/PCDFs) resulting from PVDC combustion was no higher than that from heating air alone, and thus far below the levels which resulted from PVDC combustion at 750 degrees C or lower. The results provide the first laboratory confirmation of the relation between PVDC incineration temperature and PCDD/PCDF formation, and of the primary importance of high temperature, turbulence for mixing between air and combustion gas, and sufficient residence time, as governing factors for the minimization of PCDD/PCDF formation in municipal solid waste incinerators.

Method for the simultaneous determination of losartan and its major metabolite, EXP-3174, in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry.

A liquid chromatography-electrospray ionization tandem mass spectrometric method was developed for the simultaneous determination of losartan and its major active metabolite, EXP-3174, in human plasma. The two analytes and the internal standard (DuP-167) were extracted from plasma under acidic conditions by using solid-phase extraction cartridges containing a sorbent of copolymer, poly(divinylbenzene-co-N-vinylpyrrolidone). The analytes were separated by LC equipped with a reversed-phase C18 column, and introduced into the mass spectrometer via the electrospray ion source with pneumatically-assisted nebulization. For LC-MS-MS samples, an isocratic mobile phase consisting of [0.1% triethylamine-0.1% acetic acid (pH 7.1)]-acetonitorile (65:35, v/v) was used, and the assay was monitored for the negative fragment ions of the analytes. The method demonstrated linearity from 1 to 1000 ng/ml for both losartan and EXP-3174. The limit of quantification for both compounds in plasma was 1 ng/ml. This assay method may be useful for the measurement of levels of the two compounds in clinical studies of losartan.

How vertebrate and invertebrate visual pigments differ in their mechanism of photoactivation.

In vertebrate visual pigments, a glutamic acid serves as a negative counterion to the positively charged chromophore, a protonated Schiff base of retinal. When photoisomerization leads to the Schiff base deprotonating, the anionic glutamic acid becomes protonated, forming a neutral species that activates the visual cascade. We show that in octopus rhodopsin, the glutamic acid has no anionic counterpart. Thus, the "counterion" is already neutral, so no protonated form of an initially anionic group needs to be created to activate. This helps to explain another observation-that the active photoproduct of octopus rhodopsin can be formed without its Schiff base deprotonating. In this sense, the mechanism of light activation of octopus rhodopsin is simpler than for vertebrates, because it eliminates one of the steps required for vertebrate rhodopsins to achieve their activating state.