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.

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.

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.

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.

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.

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.

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.

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.

Compression of the common bile duct by the posterosuperior pancreaticoduodenal artery: case report.

We describe a unique case of a patient who presented with a linear, transverse filling defect in the common bile duct, detected by cholangiography, that was caused by the posterosuperior pancreaticoduodenal artery compressing the common bile duct associated with gallstones. We believe this case to be the first of its kind reported in the literature.

Detection of identical JC virus DNA sequences in both human kidneys.

We studied JC virus (JCV) DNA sequence diversity among kidneys derived from cadavers with various causes of death. The 610-bp JCV DNA sequences we evaluated were identical not only among specimens derived from the same kidney but also among those derived from both kidneys of the same cadaver. Because the left and right kidneys are anatomically independent, our findings suggest that the viremia that has been proposed to occur after primary infection distributes the same JCV strain to both kidneys.

Clinical significance of AIB1 expression in human breast cancer.

AIB1 (amplified in breast cancer 1) is a member of the steroid receptor coactivator family and is a key factor in enhancing estrogen-dependent transcription. To evaluate the clinical significance of AIB1 in breast cancer, we performed Southern blot analysis of the AIB1 gene on 124 human breast cancer tissues. We also performed reverse transcription-polymerase chain reaction and semi-quantitative analysis of AIB1 mRNA expression on 58 of the tissues, and immunohistochemical detection of AIB1 protein on 115 of the tissues. On Southern blot analysis, the AIB1 gene was amplified in only two of the 124 breast cancer cases. On semi-quantitative analysis, the relative expression level of AIB1 normalized to that of GAPDH varied from 0.247 to 7.721 (median = 0.94), and was not correlated with any clinico-pathological factors. Although most of the breast cancer cells revealed cytoplasmic staining of AIB1, only 16% (18 in 115) showed nuclear staining of AIB1 protein. AIB1 nuclear expression was correlated with positivity for estrogen receptor alpha (P = 0.022). Those patients with tumor samples that showed nuclear staining of AIB1 tended to be successfully treated by endocrine therapy in comparison with those who did not show nuclear staining of AIB1. In conclusion, AIB1 nuclear expression was correlated with the estrogen receptor alpha status, and patients with AIB1 nuclear expression tended to be successfully treated by hormonal therapy.

Endostatin gene therapy on murine lung metastases model utilizing cationic vector-mediated intravenous gene delivery.

Tumors require ongoing angiogenesis to support their growth. Inhibition of angiogenesis by production of antiangiogenic factors should be a viable approach for cancer gene therapy. In this study, we investigated whether intravenous administration of endostatin gene complexed with a cationic vector (GL67/DOPE or PEI22K) could inhibit the development of lung tumors in mice injected i.v. with NFSa Y83 fibrosarcoma cells (5 x 10(5)) which frequently form lung metastasis. mRNA and protein of the transfected gene were produced in the lung and other organs of the transfected mice as assessed by immunohistochemistry, Western blotting and reverse transcription-polymerase chain reaction. Single intravenous injection of the endostatin gene (60 microg) complexed with either GL67/DOPE or PEI22K on day 3 or day 7 after fibrosarcoma cell inoculation significantly inhibited tumor formation in the lung as evidenced by the reduced number of lung tumors and lung weight, and prolonged survival of the endostatin gene-transfected mice compared with control mice. These findings suggested that the endostatin gene therapy, using cationic vector-mediated intravenous gene transfer, might be a feasible strategy for organ-targeted prevention and regulation of possible disseminated cancers.

JC virus genotyping offers a new means of tracing the origins of unidentified cadavers.

There has been no reliable means of tracing the origins of unidentified cadavers but the recent finding that JC virus (JCV) can serve as a means of elucidating human migrations suggested that this virus may also be useful to trace the origins of unidentified cadavers. DNA samples extracted from renal tissue and urine were used as the template for PCR amplification of a 610 bp region (IG region) of the viral genome. We detected JCV DNA in 45% of the renal samples and in 33% of the urine samples and was detectable even 10 days after death. The sequences of the amplified IG regions could be used to determine the genotypes. We conclude that the JC virus genotype is a new marker useful for tracing the origins of unidentified cadavers.

Evaluation of oestrogen receptor beta wild-type and variant protein expression, and relationship with clinicopathological factors in breast cancers.

We addressed the clinicopathological significance of the oestrogen receptor (ER) beta protein, including an ERbeta variant, ERbetacx, in normal human breast and breast cancer. The reverse transcriptase-polymerase chain reaction (RT-PCR) showed that wild-type ERbeta (ERbetaw) mRNA expression was higher in normal than cancer tissues, and that ERbetacx mRNA was higher in cancer than in normal tissues. Immunohistochemistry of 22 normal breast tissues and 57 breast cancers was performed with three different ERbeta antibodies and one ERbetacx antibody. All normal breast samples showed staining with the three ERbeta antibodies, suggesting that ERbetaw might have a physiological role in oestrogen signalling in the normal breast. In breast cancer, expression of the ERbetaw protein correlated well with the expression of the ERalpha and progesterone receptor (PgR), as well as histological grade (HG), and tended to indicate a better prognosis than when ERbetaw was absent. Thirty-one (54%) breast cancer samples contained ERbetacx, whereas the corresponding tissue for normal breast samples stained positive in only two (9%).