Evaluation of an amplicon-based custom gene panel for the diagnosis of hereditary tumors.

Genetic testing based on next-generation sequencing (NGS) analysis has recently been used to diagnose hereditary diseases. In this study, we explored the usefulness of our custom amplicon panel that targeted 23 genes related to hereditary tumors given in the American College of Medical Genetics and Genomics recommendations. We applied our custom NGS panel to samples from 12 patients previously diagnosed by Sanger sequencing as having the diseases or diagnosed clinically by meeting the diagnostic criteria in this study. Our gene panel not only successfully identified all variants detected by Sanger sequencing but also identified previously unrecognized variants that resulted in confirmation of the disease, or even in the revision of the diagnosis. For instance, a patient identified with an SDHD gene mutation actually had von Hippel-Lindau (VHL) syndrome, as determined by the presence of a pathogenic VHL gene variant. We also identified false-positive results that were generated by amplification of genome regions that are not intended to be investigated. In conclusion, NGS-based amplicon sequencing is a highly effective method to detect germline variants, as long as they are also carefully reviewed by manual inspection.

Xenotransplantation-the current status and prospects.

Introduction: There is a continuing worldwide shortage of organs from deceased human donors for transplantation into patients with end-stage organ failure. Genetically engineered pigs could resolve this problem, and could also provide tissues and cells for the treatment of conditions such as diabetes, Parkinson's disease and corneal blindness. Sources of data: The current literature has been reviewed. Areas of agreement: The pathobiologic barriers are now largely defined. Research progress has advanced through the increasing availability of genetically engineered pigs and novel immunosuppressive agents. Life-supporting pig kidneys and islets have functioned for months or years in nonhuman primates. Areas of controversy: The potential risk of transfer of a pig infectious microorganism to the recipient continues to be debated. Growing points: Increased attention is being paid to selection of patients for initial clinical trials. Areas timely for developing research: Most of the advances required to justify a clinical trial have now been met.

Bringing Home The Bacon: Update on The State of Kidney Xenotransplantation.

BACKGROUND: There is a continuing critical shortage of organs from deceased human donors for transplantation, particularly for patients awaiting kidney transplantation. Efforts are being made to resolve the donor kidney shortage by the transplantation of kidneys from genetically-engineered pigs. SUMMARY: This review outlines the pathobiological barriers to pig organ xenotransplantation in primates, which include (i) antibody-dependent complement-mediated rejection, (ii) a T cell-mediated elicited antibody and cellular response, (iii) coagulation dysregulation between pigs and primates, and (iv) a persistent inflammatory response. As a result of increasing genetic manipulation of the pig and the introduction of novel immunosuppressive agents, pig kidney graft survival has increased from minutes to months, and even to >1 year in some cases. Aspects of the selection of the patients for a first clinical trial are discussed. Although there would appear to be some cross-reactivity between anti-human leukocyte antigen (HLA) antibodies and swine leukocyte antigens expressed in pigs, some HLA-sensitized patients will be at no disadvantage if they receive a pig kidney. Furthermore, the current limited evidence is that, even if the patient becomes sensitized to pig antigens (after a pig organ transplant), this would not be detrimental to a subsequent allotransplant. The potential risk of infection with a pig microorganism, and the function of a pig kidney in a primate are also discussed. Key Message: The recent encouraging results of pig kidney transplantation in nonhuman primates suggest the likelihood of a successful (and safe) initial clinical trial, with graft survival for months or possibly years.

Ethinylestradiol is beneficial for postmenopausal patients with heavily pre-treated metastatic breast cancer after prior aromatase inhibitor treatment: a prospective study.

BACKGROUND: Oestrogens usually stimulate the progression of oestrogen receptor (ER)-positive breast cancer. Paradoxically, high-dose oestrogens suppress the growth of these tumours in certain circumstances. METHODS: We prospectively examined the efficacy and safety of ethinylestradiol treatment (3 mg per day oral) in postmenopausal patients with advanced or recurrent ER-positive breast cancer who had previously received endocrine therapies, especially those with resistance to aromatase inhibitors. RESULTS: Eighteen patients were enrolled with the median age of 63 years and the mean observation time of 9.2 months. Three cases withdrew within 1 week due to oestrogen flare reactions with nausea, fatigue and muscle-skeletal pain. The response rate was 50% (9 out of 18), and the clinical benefit rate was 56% (10 out of 18). The stable disease (<6 months) was 17% (3 out of 18) and another 2 cases were judged as progressive disease. Time-to-treatment failure including 2 on treatment was a median of 5.6 months (range 0.1 to 14.5(+)). Although vaginal bleeding or endometrial thickening was observed in patients receiving long-term treatment, there were no severe adverse events, such as deep venous thrombosis or other malignancies. CONCLUSION: Although the mechanism of this treatment has not been fully understood, our data may contribute to change the common view of late-stage endocrine therapy.

Estrogen receptor-positive breast cancer in Japanese women: trends in incidence, characteristics, and prognosis.

BACKGROUND: The incidence of breast cancer in Japanese women has doubled in all age groups over the past two decades. PATIENTS AND METHODS: We examined the characteristics of the tumors treated in three time periods between 1982 and 2010. Estrogen receptor (ER), progesterone receptor (PgR) and HER2 status were assessed by immunohistochemistry. Correlation of hormone receptor levels with clinicopathological factors and prognosis was analyzed in ER-positive, HER2-negative breast cancer in two age groups (≤50 years versus >50 years). RESULTS: The frequency of ER-positive breast cancer in women aged 50 years or younger increased greatly over the interval studied (1982-1991: 52.5%, 1992-2001: 72.6%, 2002-2010: 87.1%, P < 0.0001). The frequency of ER-positive tumors also significantly increased in women over 50 years of age (1982-1991: 69.4%, 1992-2001: 73.3%, 2002-2010: 78.6%, P = 0.029). In ER-positive, HER2-negative breast cancer, tumor grade was negatively correlated with expression levels of ER and PgR. Prognosis for patients with ER-positive, HER2-negative disease significantly improved over time, due to advances in adjuvant therapies. CONCLUSION: It is necessary to establish risk factors, both genetic and environmental, capable of predicting the risk of ER-positive breast cancer and thus enable the efficient selection of candidates for hormone receptor-targeted chemoprevention.

A phase II multi-center study of triple therapy with paclitaxel, S-1 and cisplatin in patients with advanced gastric cancer.

OBJECTIVES: To carry out a phase II multi-center study on the efficacy and safety of triple combination therapy with paclitaxel, S-1, and cisplatin in patients with unresectable/metastatic gastric cancer. METHODS: A total of 63 patients from 8 institutions were included in this study. Paclitaxel (160 mg/m²) was administered by infusion for 3 h on the first day. S-1 (70 mg/m²/day) was administered orally for 14 consecutive days from the first day. Cisplatin (60 mg/m²) was administered intravenously over 24 h on day 14 of every 28-day cycle. RESULTS: All 63 patients were assessed for clinical efficacy and safety. A total of 259 cycles of treatment were administered (median 4, range 1-10). Grade 3-4 toxicities included neutropenia in 30.2%, thrombocytopenia in 12.7%, and anemia in 11.1%. There was no grade 3-4 non-hematological toxicity or treatment-related death. Complete response was observed in 6 patients and partial response in 34 patients. The overall response rate was 63.5%. The median progression-free survival and response duration were 8.0 and 8.8 months, respectively, and median survival time was 15 months. CONCLUSIONS: Triple combination therapy with paclitaxel, S-1, and cisplatin showed promising safety and efficacy profiles with the potential to become a standard regimen for unresectable/metastatic gastric cancer.

The treatment of uterine leiomyosarcoma: clinical outcomes of 18 cases and the effectiveness of chemotherapy.

PURPOSE: To investigate clinical outcomes with respect to the effectiveness of chemotherapy in the treatment of uterine leiomyosarcoma. METHODS: Study subjects were 18 patients with uterine leiomyosarcoma treated surgically at our hospital between February 1986 and December 2007. A chemotherapy regimen that combined ifosfamide, epirubicine, and cisplatin (IEP) was used as the main first-line chemotherapy. RESULTS: FIGO disease stages were as follows: Stage I (n = 11), Stage II (n = 1), Stage III (n = 3), Stage IV (n = 3). Five-year overall survival of patients with Stage I-III disease was 65.3% (95% CI: 46.1-92.4%). None of patients with Stage IV disease survived for more than two years. Of seven patients who suffered advanced or recurrent disease, six received IEP; the response rate was 50%, one complete response and two partial responses. CONCLUSIONS: The combination of surgery and chemotherapy seems to be an acceptable treatment for uterine leiomyosarcoma. IEP may be an active regimen for this aggressive disease.

Three dose regimens of fulvestrant in postmenopausal Japanese women with advanced breast cancer: results from a double-blind, phase II comparative study (FINDER1).

BACKGROUND: FINDER1 compared efficacy, tolerability and pharmacokinetics (PK) of three fulvestrant dose regimens in postmenopausal Japanese women with estrogen receptor (ER)-positive locally advanced/metastatic breast cancer recurring or progressing after prior endocrine therapy. PATIENTS AND METHODS: The primary end point of this randomised, multicentre, phase II study was objective response rate (ORR) and the secondary end points included time to progression (TTP), clinical benefit rate (CBR), PK profiles and tolerability. Postmenopausal women with ER-positive advanced breast cancer were randomised to 28-day cycles of fulvestrant approved dose (AD), loading dose (LD) or high dose (HD) until disease progression. RESULTS: Hundred and forty-three patients (median age 61 years) received fulvestrant AD (n = 45), LD (n = 51) or HD (n = 47). ORR was similar across dose regimens: 11.1%, 17.6% and 10.6% for AD, LD and HD, respectively, with overlapping confidence intervals. TTP and CBR were also similar between groups (median TTP: 6.0, 7.5 and 6.0 months, respectively; CBR: 42.2%, 54.9% and 46.8% for AD, LD and HD, respectively). C(max) and area under the plasma concentration-time curve were dose proportional and PK steady state was reached earlier with LD and HD than with AD. All three doses were well tolerated, with a similar adverse-event profile and no emerging safety concerns. CONCLUSION: Fulvestrant AD, LD and HD had similar efficacy and tolerability profiles in postmenopausal Japanese women with ER-positive advanced breast cancer.

A new fast algorithm to achieve the dose uniformity around high dose rate brachytherapy stepping source using Tikhonov regularization.

The dose optimization algorithm based on anatomical points is developed to produce rapidly uniform doses over target distances generated on the target volume edges in high-dose-rate (HDR) brachytherapy stepping source application for a treatment length of 6 cm. Monte Carlo modeling of the 60Co HDR brachytherapy source and the surrounding medium were performed using PHITS code. The source dwell times were optimized using Tikhonov regularization in order to obtain uniform dose distribution at the anatomical points located at predefined target distances. The computed dose rates at distances from 0.25 up to 20 cm away from the source were first verified with the literature data sets. Then, the simulation results of the optimization process were compared to the calculations of commercial treatment planning system (TPS) SagiPlan. As a result, the dose uniformity was observed in the isodose curves at the target distances of 10 and 15 mm of the treatment length and the prescribed dose achieved the anatomical points uniformly. The algorithm developed in the present study can be applied for achieving the dose uniformity around the brachytherapy stepping source as a quicker tool for different treatment lengths and different target distances while maintaining the high quality of the treatment plans, saving time by avoiding the manual isodose shaping and then better suitable treatment for patients.

Midkine as a prognostic biomarker in oral squamous cell carcinoma.

The aim of this study was to evaluate serum midkine (S-MK) concentrations as a prognostic tumour marker in oral squamous cell carcinoma (OSCC). We measured S-MK concentrations in patients with OSCC and healthy volunteers. In addition, we performed real-time quantitative reverse transcription-PCR analysis and immunohistochemistry with fresh tumour samples. To determine whether S-MK concentrations have prognostic value, we performed survival analyses with clinical information by using the log-rank test. Serum midkine concentrations were significantly higher in patients with OSCC than in healthy controls (P<0.001). Serum midkine concentrations were also significantly increased in early-stage OSCC compared with those of healthy individuals (P<0.001). In addition, immunohistochemistry allowed identification of overexpressed MK protein in OSCC tissues. MK mRNA showed higher expression in OSCC samples compared with normal mucosal samples. Patients in high S-MK groups showed a significantly lower 5-year survival rate compared with patients in low S-MK groups (P<0.05). The increased S-MK concentrations in early-stage OSCC were strongly associated with poor survival. Serum midkine concentrations may thus be a useful marker not only for cancer screening but also for predicting prognosis of OSCC patients.

Experimental and theoretical study of the neutron dose produced by carbon ion therapy beams.

High-energy (12)C ions offer favourable conditions for the treatment of deep-seated local tumours. Several facilities for the heavy ion therapy are planned or under construction, for example the new clinical ion-therapy unit HIT at the Radiological University Clinics in Heidelberg. In order to improve existing treatment planning models, it is essential to evaluate the secondary fragment production and to include these contributions to the therapy dose with higher accuracy. Secondary neutrons are most abundantly produced in the reactions between (12)C beams and tissues. The dose contribution to tissues by a neutron is fairly small compared with the projectile and the other charged fragments due to no ionisation and the small reaction cross-sections; however, it distributes in a considerably wider region beyond the bragg-peak because of the strong penetrability. Systematic data on energy spectra and doses of secondary neutrons produced by (12)C beams using water targets of different thicknesses for various detection angles have therefore been measured in this study at GSI Darmstadt.

Stat5 expression predicts response to endocrine therapy and improves survival in estrogen receptor-positive breast cancer.

Constitutively activated signal transducers and activators of transcription (Stats), in particular Stat3 and Stat5, have been demonstrated to directly contribute to oncogenesis by stimulating cell proliferation and preventing apoptosis in various cancers. Stat3 is essential in mammary gland epithelial cell apoptosis and involution, whereas Stat5 is well established as a key factor in mammary epithelial cell growth and differentiation. Crosstalk between Stats and estrogen receptor (ER) has been demonstrated by several laboratories and we have focused on the role of Stat5 in ER-positive breast cancer. Using immunohistochemical techniques, we examined the expression of Stat3 and Stat5 in 517 human breast cancer tissues and analyzed their significance for prognosis and prediction of response to endocrine therapy. Stat5 expression was significantly correlated with histological grade (P<0.0001), ER (P=0.02), and progesterone receptor (P=0.026) expression. There was no difference between Stat3 expression and clinicopathological factors. In 346 patients with ER-positive breast cancer, patients with Stat5 positive tumors had significantly increased overall survival (P=0.0009) in multivariate analysis. There were 70 patients who received endocrine therapy as first-line treatment for metastatic breast cancer at relapse. The patients whose primary breast tumors were Stat5 positive, had significantly better response to endocrine therapy (P=0.04), and longer survival after relapse (P=0.0003), than those whose tumors were Stat5 negative. The present study demonstrates for the first time that Stat5 is a predictive factor for endocrine therapy response and a strong prognostic molecular marker in ER-positive breast cancer. Our data suggest that the expression of Stat5 is helpful in selecting patients who may benefit from endocrine therapy.

Experimental fragmentation studies with 12C therapy beams.

High-energy beams of (12)C ions in the range of 80-430 MeV u(-1) delivered by the heavy-ion synchrotron SIS-18 are used for radiotherapy of deep-seated localized tumors at the treatment unit at GSI Darmstadt. In order to improve the physical database, the fragmentation characteristics along the penetration path in tissue were investigated experimentally by using a water phantom as tissue-equivalent absorber. Measurements were performed at specific energies of 200 and 400 MeV u(-1) of the incident (12)C ions and at six different depths before and behind the Bragg peak. Secondary fragments with nuclear charges Z(f) = 1-5 were identified by scintillation detectors using DeltaE-E and time-of-flight techniques. The preliminary results include energy- and angular distributions, fragment yields, build-up curves and attenuation of the primary carbon projectiles.

Morphological, biological, and biochemical characteristics of a benign human trichilemmoma cell line in vivo and in vitro.

A cell line of a benign tumor, trichilemmoma, was established in vitro and has been maintained in culture for 1.5 years with more than 30 passages. Plating efficiency was less than 0.1%, and population doubling time was 10 days. Saturation density was 10(5) cells/sq cm at the time of a monolayer with 98% cell viability. Ultrastructurally, tissue-cultured trichilemmoma cells showed desmosome-tonofilament complexes at cell-to-cell junctions. The tissue-cultured cells synthesized abundant glycogen (50 to 100 microgram/10(6) cells) e was 10 days. Saturation density was 10(5) cells/sq cm at the time of a monolayer with 98% cell viability. Ultrastructurally, tissue-cultured trichilemmoma cells showed desmosome-tonofilament complexes at cell-to-cell junctions. The tissue-cultured cells synthesized abundant glycogen (50 to 100 microgram/10(6) cells) e was 10 days. Saturation density was 10(5) cells/sq cm at the time of a monolayer with 98% cell viability. Ultrastructurally, tissue-cultured trichilemmoma cells showed desmosome-tonofilament complexes at cell-to-cell junctions. The tissue-cultured cells synthesized abundant glycogen (50 to 100 microgram/10(6) cells) as observed in vivo. Gas chromatographic analysis revealed that extracted glycogen was composed of glucose alone. Chromosome analyses with trypsin-Giemsa banding showed an abnormal karyotype with hypodiploid modal numbers of 44 and 45. There were four marker chromosomes observed in 100% of cells in 100 metaphase cells examined. Cells did not grow on fibroblast monolayers or in soft agar in vitro but did induce tumors in athymic nude mice (12 of 15) after the s.c. injection of tissue-cultured cells (2.5 x 10(6) to 4.5 x 10(7) cells/mouse). The histological characteristics of the tumors in nude mice were similar to those of the original tumor. This is the first time, to our knowledge, that a benign human tumor cell line has been established in vitro which can induce tumors in nude mice.

Suppression of ING1 expression in sporadic breast cancer.

Down regulation of the ING1 candidate tumour suppressor promotes growth in soft agar and focus formation in vitro and tumour formation in vivo. ING1 encodes a nuclear, cell cycle-regulated protein, overexpression of which efficiently blocks cell growth and is capable of inducing apoptosis in different experimental systems. Here we present the first report of ING1 mutation and expression analysis in a total of 452 cancer samples. One germline missense alteration and three germline silent alterations were detected in 377 primary breast cancers while marked (2 - 10-fold) decreases in ING1 mRNA expression were seen in 44% of primary breast cancers and in ten of ten breast cancer cell lines examined. Furthermore, the majority of breast cancers (58%) showing decreased ING1 expression had metastasized to regional lymph nodes whereas only 9% of cancers with elevated ING1 expression, compared to adjacent normal tissues, were metastatic. Thus, ING1 mutation is very rare in breast or ovarian cancers, however, repression of ING1 expression frequently accompanies tumour development of breast cancer.

The use of mannan-Sepharose 4B affinity chromatography for the purification of endo-beta-N-acetylglucosaminidase from Bacillus alvei.

In order to facilitate the isolation of endo-beta-N-acetylglucosaminidase for the structural analysis of glycoconjugates, we have isolated a strain of Bacillus alvei which produces a high level of endo-beta-N-acetylglucosaminidase. We have also devised a simple procedure for the purification of endo-beta-N-acetylglucosaminidase from B. alvei using mannan-Sepharose affinity chromatography. By using this method, endo-beta-N-acetylglucosaminidase was purified 3300-fold with 85% yield from the crude enzyme obtained by ammonium sulfate precipitation of the culture medium. The molecular weight of this enzyme was estimated to be about 66 000 by gel filtration. When using (Man)6(GlcNAc)2-Asn-Dns as substrate, the optimal activity occurs at pH 6.5 with Km of 1.9 mM. The action of endo-beta-N-acetylglucosaminidase toward several glycopeptides was also studied.

Identification of cis-9,10-methylenehexadecanoic acid in submitochondrial particles of bovine heart.

Submitochondrial particles of bovine heart were hydrolyzed by phospholipase A2 and the products were analyzed by liquid chromatography electrospray ionization-mass spectrometry. We found a fatty acid with a molecular mass of 268 Da and a retention time longer than that of linoleic acid. Next, we synthesized organically cis-9,10-methylenehexadecanoic acid, which has a molecular mass similar to that of the extracted fatty acid, and characterized its high performance liquid chromatography and gas chromatography-mass spectrometry profiles. Using these data we were able to identify endogenous cis-9,10-methylenehexadecanoic acid in rat and human heart and liver tissues that had been hydrolyzed by phospholipase A2. This fatty acid was not detected in tissue extracts that had not been hydrolyzed by phospholipase A2. Similar amounts of cis-9, 10-methylenehexadecanoic acid were measured in tissue extracts after total hydrolysis. These results suggest that cis-9, 10-methylenehexadecanoic acid is a fatty acid component, in the sn-2 position, of phospholipids in some mammalian tissue.

Formation of leukotoxin (9,10-epoxy-12-octadecenoic acid) during the autoxidation of phospholipids promoted by hemoproteins.

Myoglobin (Mb) and cytochrome c (Cyt c) are known to promote lipid peroxidation when mixed with certain types of phospholipids. In the presence of phospholipids such as cardiolipin (CL), ferrous Mb and Cyt c were converted to ferric hemoproteins, and autoxidation of the phospholipids and the oxidation of free linoleic acid (LA) added to the reaction mixture were observed. When the reaction mixture comprising 0.01 mM Cyt c, 0.2 mM CL and 0.1 mM LA was incubated, 92.7% of LA was consumed, and the LA products included 2.49 microM 9,10-epoxy-12-octadecenoic acid (leukotoxin) and its isomer which are potent inhibitors of mitochondrial respiration and have toxic effects on cardiac function. Hemoglobin (Hb) could promote almost no lipid peroxidation in the presence of any kinds of phospholipids. The experiments using some scavengers of active oxygen species revealed that tocopherol and ascorbic acid could strongly reduced LA oxidation caused by Cyt c or Mb. As LTx production was also observed when LA was mixed with Fe2+, LTx may be a common product where non-enzymatic lipid peroxidation occurs.

A phase II multicentric trial of S-1 combined with 24 h-infusion of cisplatin in patients with advanced gastric cancer.

BACKGROUND: The aim of this multicentric trial was to determine the clinical toxicities and antitumor effects of a chemotherapy regimen of S-1 combined with cisplatin in patients with inoperable locally or metastatic advanced gastric cancer. PATIENTS AND METHODS: Forty-two patients were entered into the study. S-1 (80 mg/m2) was administered orally daily for 14 consecutive days and 24-h infusion of cisplatin (70 mg/m2) was administered on day 8 of every 28-day cycle. RESULTS: The overall response rate was 50% and complete response rate was 5%. The most common adverse event was leucopenia, which occurred with grade 3 in 7 patients (16.6%) and grade 4 in 2 patients (4.8%). Non-hematological adverse events were generally mild. The median survival time was 342 days. The 2-year survival rate was 22.9%. CONCLUSION: This combination chemotherapy is active, convenient and well tolerated in patients with high-grade advanced gastric cancer.

Simulations of an accelerator-based shielding experiment using the particle and heavy-ion transport code system PHITS.

In order to estimate the biological effects of HZE particles, an accurate knowledge of the physics of interaction of HZE particles is necessary. Since the heavy ion transport problem is a complex one, there is a need for both experimental and theoretical studies to develop accurate transport models. RIST and JAERI (Japan), GSI (Germany) and Chalmers (Sweden) are therefore currently developing and bench marking the General-Purpose Particle and Heavy-Ion Transport code System (PHITS), which is based on the NMTC and MCNP for nucleon/meson and neutron transport respectively, and the JAM hadron cascade model. PHITS uses JAERI Quantum Molecular Dynamics (JQMD) and the Generalized Evaporation Model (GEM) for calculations of fission and evaporation processes, a model developed at NASA Langley for calculation of total reaction cross sections, and the SPAR model for stopping power calculations. The future development of PHITS includes better parameterization in the JQMD model used for the nucleus-nucleus reactions, and improvement of the models used for calculating total reaction cross sections, and addition of routines for calculating elastic scattering of heavy ions, and inclusion of radioactivity and burn up processes. As a part of an extensive bench marking of PHITS, we have compared energy spectra of secondary neutrons created by reactions of HZE particles with different targets, with thicknesses ranging from <1 to 200 cm. We have also compared simulated and measured spatial, fluence and depth-dose distributions from different high energy heavy ion reactions. In this paper, we report simulations of an accelerator-based shielding experiment, in which a beam of 1 GeV/n Fe-ions has passed through thin slabs of polyethylene, Al, and Pb at an acceptance angle up to 4 degrees.