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.

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.

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.

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.

Initial in vivo experience of pig artery patch transplantation in baboons using mutant MHC (CIITA-DN) pigs.

BACKGROUND: In the pig-to-nonimmunosuppressed baboon artery patch model, a graft from an α1,3-galactosyltransferase gene-knockout pig transgenic for human CD46 (GTKO/CD46) induces a significant adaptive immune response (elicited anti-pig antibody response, increase in T cell proliferation on MLR, cellular infiltration of the graft), which is effectively prevented by anti-CD154mAb-based therapy. METHODS: As anti-CD154mAb is currently not clinically applicable, we evaluated whether it could be replaced by CD28/B7 pathway blockade or by blockade of both pathways (using belatacept + anti-CD40mAb [2C10R4]). We further investigated whether a patch from a GTKO/CD46 pig with a mutant human MHC class II transactivator (CIITA-DN) gene would allow reduction in the immunosuppressive therapy administered. RESULTS: When grafts from GTKO/CD46 pigs were transplanted with blockade of both pathways, a minimal or insignificant adaptive response was documented. When a GTKO/CD46/CIITA-DN graft was transplanted, but no immunosuppressive therapy was administered, a marked adaptive response was documented. In the presence of CD28/B7 pathway blockade (abatacept or belatacept), there was a weak adaptive response that was diminished when compared with that to a GTKO/CD46 graft. Blockade of both pathways prevented an adaptive response. CONCLUSION: Although expression of the mutant MHC CIITA-DN gene was associated with a reduced adaptive immune response when immunosuppressive therapy was inadequate, when blockade of both the CD40/CD154 and CD28/B7 pathways was present, the response even to a GTKO/CD46 graft was suppressed. This was confirmed after GTKO/CD46 heart transplantation in baboons.

Evaluation of a personal identification method using the fusion function of CT images and dental radiographs.

OBJECTIVES: We applied the fusion function of dental analysis software to examine whether the anatomical relationships of tooth roots when compared between reconstructed post-mortem CT (PMCT) and dental radiographs can aid dental identification. METHODS: One PMCT image taken from a cadaver (43-year-old male; Cadaver 1) was compared with 64 digital dental radiographs of the left and right upper and lower molars from Cadaver 1 and 30 other cadavers. Five corresponding anatomical reference points were marked on each image. After adjusting the angle and magnification using the fusion function, the automatically calculated error in pixels was determined five times for each of four sites on the images. RESULTS: Comparison of the PMCT image with the dental radiographs from the other cadavers revealed obvious discrepancies in the anatomical positioning of the teeth. When t-tests were applied to the data from any of the four sites, the error in pixels was found to be significantly smaller (p < 0.001) between Cadaver 1 and the other cadaver images. The average error in pixels between the PMCT and dental radiographs was smaller in the lower jaw than in the upper jaw. CONCLUSIONS: This method uses corresponding reference points on two images to calculate the error between the regions that contain all points. This feature also makes it possible to compare images taken with different modalities. The demand for a dental identification method involving PMCT is likely to increase, and we expect that the accuracy of dental identification will improve by using radiological images.

Experimental study of nuclear fragmentation of 200 and 400 MeV/u (12)C ions in water for applications in particle therapy.

Carbon ion beams in the energy range of about 100-450 MeV/u offer excellent conditions for tumour therapy, in particular for the treatment of deep-seated radio-resistant tumours. Their depth-dose distribution is characterized by a low dose in the entrance channel, small lateral beam spread and an elevated biological effectiveness in the Bragg peak region. In comparison to protons the radiation field of heavier ions stopping in tissue is however more complex due to nuclear fragmentation reactions occurring along their stopping path. This results in an attenuation of the primary beam flux and a build-up of lower-Z fragments with longer ranges causing the characteristic dose tail beyond the Bragg peak. In the present work the characteristics of secondary charged particles at various depths of water were investigated experimentally using (12)C ion beams of 200 and 400 MeV/u delivered by the heavy-ion synchrotron SIS-18 at GSI Darmstadt. The nuclear charge Zf of secondary fragments was identified by combining energy loss and time-of-flight (TOF) measurements. Energy spectra and yields were recorded at lab angles of 0° - 10° and at seven different water depths corresponding to the entrance channel, the Bragg peak region and the tail of the Bragg curve.

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.

Three-dimensional visualization of composite fillings for dental identification using CT images.

OBJECTIVES: This study aimed to discriminate between enamel and composite resins by differences in Hounsfield units shown on 16 section multidetector CT (MDCT) images taken of unidentified bodies. METHODS: First, we determined the Hounsfield units of composite resins in 15 extracted human teeth. We then filled a single cavity prepared in each of the teeth with one of five different types of composite resins, and scanned the teeth using our routine post-mortem CT protocol for the head and neck. Obtained data were transferred to a radiological workstation and reconstructed. Furthermore, post-mortem CT images of the head of three unidentified bodies were reconstructed in the same manner. RESULTS: Four types of composite resins containing radio-opaque fillers showed a constant value of 4000 HU, and one radiolucent composite resin showed values in the range of 660-800 HU in the extracted teeth. Pixels at 4000 HU indicated that the composite resins were selected and visualized as three-dimensional colour images. Composite resins could be visualized on reconstructed images of the three unidentified bodies, and the sites visualized matched those noted on the forensic dental charts. CONCLUSIONS: Discriminating enamel and composite resins containing radio-opaque materials was difficult because of their similar Hounsfield unit values. However, we did succeed in visualizing the composite resins despite limitations of the CT scale. CT reconstructed images can contribute to dental identification, particularly in cases where it is difficult to detect composite resins on external investigation, and these images can be prepared during routine dental identification work.

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.

Evidence-based therapy according to the guideline for gastric ulcers is cost-effective in Japan.

Helicobacter pylori (H. pylori) infection is a major cause of gastric ulcers (GU) and eradication of the infection controls the ulcer with no requirement for maintenance therapy. In Japan, an evidence-based guideline (GL) was first published in 2003 (1(st) version) and then again in 2007 (2(nd) version) with a minor revision under support of the Japanese Ministry of Health, Labor and Welfare (GUGLJ). Adherence to its standards is high, estimated at 80%. GU patients aged 18 or older with active ulcers at the time of diagnosis by an endoscopic examination at National Hospital Organization (NHO) hospitals of Japan were enrolled between September 2004 and April 2005. Subjective and endoscopic outcome, medical treatments and medical costs during the following nine months were analyzed, retrospectively. As a result, 935 patients and 270 doctors in charge from 62 NHO hospitals were analyzed. Among H. pylori-positive GU patients, the endoscopic recurrence rate of 24 patients with failure of eradication was 29.2%, which was significantly higher than 8.8% of 194 patients with successful eradication. Successful eradication of H. pylori resulted in significantly lower endoscopic recurrence rates for GU patients either with or without administration of non-steroidal anti-inflammatory drugs (NSAID). GUGLJ adherence scores were significantly related to the specialty or knowledge on the GUGLJ of doctors in charge, and the total medical cost consumed. These results suggest that the therapy of GU along with an evidence-based GL is essential to implement cost-effective treatment and the GI experts or the doctors that understand the GUGLJ very well should perform it.

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.

Analysis of the effect of structural materials in a wall-less tissue-equivalent proportional counter irradiated by 290 MeV u(-1) carbon beam.

Effects of structural materials in a wall-less tissue-equivalent proportional counter were evaluated based on the calculation of energy deposits by EGS5 and the measurement of lineal energy distributions using 290 MeV u(-1) carbon beams. It is found that the correction of measured data based on simulation is necessary for understanding the energy deposition spectra in the homogeneous condition in tissues.

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.

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.

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.

Bacterium organizes hierarchical amorphous structure in microbial cellulose.

A pellicle, a gel film of microbial cellulose, is a supermolecular system containing 99% of water by weight, which is closely related to an amorphous structure in it. Using ultra-small-angle neutron scattering, in order to cover over a wide range of length scales from nm to 10 microm, we examined the hierarchical amorphous structure in the microbial cellulose, which is synthesized by a bacterium (Acetobacter xylinum). The microbial cellulose swollen by water shows small-angle scattering that obeys a power law q -behavior according to q -alpha as a function of the magnitude of the scattering vector q . The power law, determined by scattering, is attributed to a mass fractal due to the distribution of the center of mass for the crystallite (microfibril) in amorphous cellulose swollen by water. As q increases, alpha takes the values of 2.5, 1, and 2.35, corresponding, respectively, to a gel network composed of bundles, a bundle composed of cellulose ribbons, and concentration fluctuations in a bundle. From the mass fractal q -behavior and its length scale limits, we evaluated a volume fraction of crystallite in microbial cellulose. It was found that 90% of the cellulose bundle is occupied by amorphous cellulose containing water.

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.

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.

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.