Effect of Positive Biopsy Core Rate on Low-dose-rate Brachytherapy Outcomes in Intermediate-risk Prostate Cancer.

BACKGROUND/AIM: Intermediate-risk prostate cancer (PCa) is a highly heterogeneous disease. Although low-dose-rate brachytherapy (LDR-BT) is mainly used for low- to intermediate-risk PCa, limited reports have evaluated the detailed differences in outcomes, including differences between patients with ISUP grade group (GG) 2 and GG3 intermediate-risk PCa. This study aimed to investigate the differences in outcomes between intermediate-risk Japanese patients with GG2 and GG3 PCa who underwent LDR-BT. PATIENTS AND METHODS: This single-center retrospective study included 342 consecutive patients with intermediate-risk PCa; 232 patients with GG2 and 110 with GG3 were treated with LDR-BT at Tokushima University Hospital between July 2004 and December 2019. RESULTS: No significant difference in 5-year biochemical progression-free survival and cancer-specific survival was observed between patients with GG2 and those with GG3 (p=0.649 and p=0.633, respectively). Multivariate analysis showed that radiation doses up to 90% of the prostate volume (D90) and the percentage of positive cores were predictors of recurrence in all patients with intermediate-risk PCa. Group analyses showed that D90 was a predictor for recurrence in patients with GG2. In contrast, a high percentage of positive cores was a significant risk factor for recurrence in patients with GG3. CONCLUSION: Positive core ratios observed on prostate biopsy correlated with higher recurrence rates after LDR-BT. This indicates that the proportion of positive cores in the biopsy may be an important factor in predicting the likelihood of recurrence, especially for patients with GG3 PCa.

Prostate-specific Antigen Levels Following Brachytherapy Impact Late Biochemical Recurrence in Japanese Patients With Localized Prostate Cancer.

BACKGROUND/AIM: Evaluation of long-term outcomes is essential for the successful treatment of localized prostate cancer; however, the risk of late recurrence following brachytherapy is still not clear. This study aimed to evaluate the long-term outcomes of low-dose-rate brachytherapy (LDR-BT) for localized prostate cancer in Japanese patients and identify factors associated with late recurrence after treatment. PATIENTS AND METHODS: This single-center, cohort study included patients who underwent LDR-BT at the Tokushima University Hospital in Japan between July 2004 and January 2015; 418 patients, who were followed-up at least 7 years after LDR-BT, were included in the study. Biochemical progression free survival (bPFS) was defined according to the Phoenix definition (nadir PSA+2 ng/ml) and bPFS and cancer specific survival (CSS) were calculated using Kaplan-Meier survival curves. Univariate and multivariate analyses were performed using Cox proportional hazard regression models. RESULTS: Approximately half of the patients with PSA >0.5 ng/ml at 5 years after LDR-BT had a recurrence within the next 2 years. However, only 1.4% of the patients with a PSA ≤0.2 ng/ml at 5 years post-treatment showed tumor recurrence, including those at high risk of treatment failure according to the D'Amico classification. In multivariate analysis, PSA level at 5 years post-treatment was the only predictor of late recurrence after 7 years of treatment. CONCLUSION: PSA levels at 5 years post-treatment were associated with long-term recurrence of localized prostate cancer, which can help alleviate patient anxiety concerning prostate cancer recurrence if PSA levels remain low at 5 years after LDR-BT.

Rapid DNA Sequencing Technology Based on the Sanger Method for Bacterial Identification.

Antimicrobial resistance, a global health concern, has been increasing due to inappropriate use of antibacterial agents. To facilitate early treatment of sepsis, rapid bacterial identification is imperative to determine appropriate antibacterial agent for better therapeutic outcomes. In this study, we developed a rapid PCR method, rapid cycle sequencing, and microchip electrophoresis, which are the three elemental technologies for DNA sequencing based on the Sanger sequencing method, for bacterial identification. We achieved PCR amplification within 13 min and cycle sequencing within 14 min using a rapid thermal cycle system applying microfluidic technology. Furthermore, DNA analysis was completed in 14 min by constructing an algorithm for analyzing and performing microchip electrophoresis. Thus, the three elemental Sanger-based DNA sequencing steps were accomplished within 41 min. Development of a rapid purification process subsequent to PCR and cycle sequence using a microchip would help realize the identification of causative bacterial agents within one hour, and facilitate early treatment of sepsis.

Molecularly imprinted polymer nanogel-based fluorescence sensing of pork contamination in halal meat extracts.

Pork contamination is a serious concern for the global halal food market because many manufacturers commonly use pork instead of beef to reduce production costs. In this study, a highly sensitive fluorescent molecularly imprinted polymer nanogel (F-MIP-NG)-based sensor was developed for rapid porcine serum albumin (PSA) detection to investigate pork contamination in halal meat extracts. F-MIP-NGs were prepared via molecular imprinting and conjugation with ATTO 647N as the fluorescent reporter molecule for the post-imprinting modification (PIM) and then immobilized on gold-coated sensor chips. For achieving rapid and easy measurement, the fluorescence response was measured using a custom-made liquid handling robot equipped with a fluorescence microscope. The fluorescence response increased with increasing PSA concentration. Under optimal conditions, the F-MIP-NG-based sensors exhibited high sensitivity, a detection limit of 40 pM, a linear range of 0.25-5 nM, and excellent affinity and selectivity towards PSA, compared to potentially interfering proteins. Moreover, it was more efficient to detect beef contamination in 1 wt% pork contamination compared to the real-time polymerase chain reaction. Collectively the good analytical performance, high rates of recovery in real meat extract samples, fast detection, and a low detection limit of pork contamination (0.1 wt%) indicated the potential of the proposed sensor for detecting PSA as a marker of pork contamination in halal meat samples. The proposed sensing system based on the MIPs would open a way to establish highly sensitive and rapid sensing systems (<5 min/sample) for food analysis.

Effect of dental metal artifact conversion volume on dose distribution in head-and-neck volumetric-modulated arc therapy.

PURPOSE: During treatment planning for head-and-neck volumetric-modulated arc therapy (VMAT), manual contouring of the metal artifact area of artificial teeth is done, and the area is replaced with water computed tomography (CT) values for dose calculation. This contouring of the metal artifact areas, which is performed manually, is subject to human variability. The purpose of this study is to evaluate and analyze the effect of inter-observer variation on dose distribution. METHODS: The subjects were 25 cases of cancer of the oropharynx for which VMAT was performed. Six radiation oncologists (ROs) performed metal artifact contouring for all of the cases. Gross tumor volume, clinical target volume, planning target volume (PTV), and oral cavity were evaluated. The contouring of the six ROs was divided into two groups, small and large groups. A reference RO was determined for each group and the dose distribution was compared with those of the other radiation oncologists by gamma analysis (GA). As an additional experiment, we changed the contouring of each dental metal artifact area, creating enlarged contours (L), reduced contours (S), and undrawn contours (N) based on the contouring by the six ROs and compared these structure sets. RESULTS: The evaluation of inter-observer variation showed no significant difference between the large and small groups, and the GA pass rate was 100%. Similar results were obtained comparing structure sets L and S, but in the comparison of structure sets L and N, there were cases with pass rates below 70%. CONCLUSIONS: The results show that the artificial variability of manual artificial tooth metal artifact contouring has little effect on the dose distribution of VMAT. However, it should be noted that the dose distribution may change depending on the contouring method in cases where the overlap between PTV and metal artifact areas is large.

Flow analysis on microcasting with degassed polydimethylsiloxane micro-channels for cell patterning with cross-linked albumin.

Patterned cell culturing is one of the most useful techniques for understanding the interaction between geometric conditions surrounding cells and their behaviors. The authors previously proposed a simple method for cell patterning with an agarose gel microstructure fabricated by microcasting with a degassed polydimethylsiloxane (PDMS) mold. Although the vacuum pressure produced from the degassed PDMS can drive a highly viscous agarose solution, the influence of solution viscosity on the casting process is unknown. This study investigated the influences of micro-channel dimensions or solution viscosity on the flow of the solution in a micro-channel of a PDMS mold by both experiments and numerical simulation. It was found experimentally that the degassed PDMS mold was able to drive a solution with a viscosity under 575 mPa·s. A simulation model was developed which can well estimate the flow rate in various dimensions of micro-channels. Cross-linked albumin has low viscosity (1 mPa·s) in aqueous solution and can undergo a one-way dehydration process from solution to solid that produces cellular repellency after dehydration. A microstructure of cross-linked albumin was fabricated on a cell culture dish by the microcasting method. After cells were seeded and cultivated on the cell culture dish with the microstructure for 7 days, the cellular pattern of mouse skeletal myoblast cell line C2C12 was observed. The microcasting with cross-linked albumin solution enables preparation of patterned cell culture systems more quickly in comparison with the previous agarose gel casting, which requires a gelation process before the dehydration process.

Detection of Salmonella Enterica in Egg Yolk by PCR on a Microfluidic Disc Device Using Immunomagnetic Beads.

Salmonella enterica is a pathogenic bacterium that causes foodborne illness. One of the vehicle foods of S. enterica are chicken eggs. Efficient collection of the bacterium is necessary to detect it specifically. We developed a method to detect S. enterica by PCR on a microfluidic disc device using a fluorescent probe. Salmonella enterica cells were isolated in the microchambers on the device, followed by thermal lysis and PCR targeting with the invA gene, a gene specific to S. enterica, were observed by measurement of the fluorescent signal that resulted from gene amplification. However, the developed method was unable to discriminate viable cells from dead cells. Consequently, in this study, magnetic beads modified with anti-Salmonella antibody were utilized to detect viable Salmonella cells from egg yolk prior to PCR on the device. While using the antibody-modified beads, egg yolk components, which inhibit PCR, were removed. The collected cells were subsequently detected by PCR of the invA gene on a microfluidic disc device. This method enabled the detection of viable cells without the inhibition of PCR by any egg component. S. enterica was detected at 5.0×104 cells mL-1 or at a higher concentration of egg yolk within 6 h including the sampling time.

Clinical outcomes of hypofractionated image-guided multifocal irradiation using volumetric-modulated arc therapy for brain metastases.

Volumetric-modulated arc therapy (VMAT) can be used to design hypofractionated radiotherapy treatment plans for multiple brain metastases. The purpose of this study was to evaluate treatment outcomes of hypofractionated image-guided multifocal irradiation using VMAT (HFIGMI-VMAT) for brain metastases. From July 2012 to December 2016, 67 consecutive patients with 601 brain metastases were treated with HFIGMI-VMAT at our institution. The prescribed dose was 50 Gy to a 95% volume of the planning target volume in 10 fractions. Fifty-five of the 67 patients had non-small-cell lung cancer, and the remaining 12 had other types of cancer. The median number of brain metastases was five, and the median maximum diameter was 1.2 cm. The median duration of follow-up was 12.0 months (range, 1.9-44.8 months), and the median survival time 18.7 months. Four patients with six lesions had local recurrences. The local control rate in the 64 assessed patients was 98.4% and 95.3% at 6 and 12 months, respectively (three died before assessment). The local control rate for the 572 assessed lesions was 99.8% and 99.3% at 6 and 12 months, respectively. Thirty-nine patients developed distant brain metastases, the distant brain control rate being 59.7% and 40.5% at 6 and 12 months, respectively. Acute toxicities were generally mild (Grade 1-2). Three patients (4.5%) developed radiation necrosis requiring corticosteroid therapy. The HFIGMI-VMAT technique with flat dose delivery was well tolerated and achieved excellent local control. This technique is a promising treatment option for patients with multiple and large brain metastases.

Rapid Enzyme-linked Immunosorbent Assays for Diagnosis of Diabetes in a Compact Disc-shaped Microfluidic Device.

We have developed a compact disc (CD)-shaped microfluidic device for multiple, rapid enzyme-linked immunosorbent assays (ELISA). The device has a versatile design that can be adapted for the detection of various proteins by selecting the push-in-type reaction parts and appropriate reagents for each target. In this paper, we report the rapid quantification of insulin, adiponectin, and leptin, which can be used for the early diagnosis of diabetes, in human serum in only 16 min with our device.

Predictive Factors for Prolonged Urination Disorder After Permanent 125I Brachytherapy for Localized Prostate Cancer.

BACKGROUND: We assessed the change in LUTS after prostate brachytherapy to reveal factors for prolonged urination disorder. MATERIALS AND METHODS: Four hundred and four patients received prostate brachytherapy at our institution and were followed-up for at least 2 years. We evaluated the correlation of mean IPSS changes and clinical factors. Using multivariate analysis, we also evaluated clinical factors with potential to delay IPSS resolution. RESULTS: In cases with prostate volume more than 30 cm3, radiation dose to 90% of prostate volume (D90) more than 160 Gy, and radiation dose to 30% of the urethral volume (UD30) more than 240 Gy, mean IPSS levels were significantly higher, even 30 months after treatment. On multivariate analysis, baseline IPSS more than 8 points and D90 more than 160 Gy were significant predictors for delayed IPSS resolution. CONCLUSION: Our data suggest that higher baseline IPSS and higher D90 were predictors for prolonged urination disorder.

On-site identification of meat species in processed foods by a rapid real-time polymerase chain reaction system.

Correct labeling of foods is critical for consumers who wish to avoid a specific meat species for religious or cultural reasons. Therefore, gene-based point-of-care food analysis by real-time Polymerase Chain Reaction (PCR) is expected to contribute to the quality control in the food industry. In this study, we perform rapid identification of meat species by our portable rapid real-time PCR system, following a very simple DNA extraction method. Applying these techniques, we correctly identified beef, pork, chicken, rabbit, horse, and mutton in processed foods in 20min. Our system was sensitive enough to detect the interfusion of about 0.1% chicken egg-derived DNA in a processed food sample. Our rapid real-time PCR system is expected to contribute to the quality control in food industries because it can be applied for the identification of meat species, and future applications can expand its functionality to the detection of genetically modified organisms or mutations.

Development of an on-site rapid real-time polymerase chain reaction system and the characterization of suitable DNA polymerases for TaqMan probe technology.

On-site quantitative analyses of microorganisms (including viruses) by the polymerase chain reaction (PCR) system are significantly influencing medical and biological research. We have developed a remarkably rapid and portable real-time PCR system that is based on microfluidic approaches. Real-time PCR using TaqMan probes consists of a complex reaction. Therefore, in a rapid real-time PCR, the optimum DNA polymerase must be estimated by using actual real-time PCR conditions. In this study, we compared the performance of three DNA polymerases in actual PCR conditions using our rapid real-time PCR system. Although KAPA2G Fast HS DNA Polymerase has the highest enzymatic activity among them, SpeedSTAR HS DNA Polymerase exhibited better performance to rapidly increase the fluorescence signal in an actual real-time PCR using TaqMan probes. Furthermore, we achieved rapid detection of Escherichia coli in 7 min by using SpeedSTAR HS DNA Polymerase with the same sensitivity as that of a conventional thermal cycler.

Rapid Detection of Salmonella enterica in Food Using a Compact Disc-Shaped Device.

Rapid detection of food-borne pathogens is essential to public health and the food industry. Although the conventional culture method is highly sensitive, it takes at least a few days to detect food-borne pathogens. Even though polymerase chain reaction (PCR) can detect food-borne pathogens in a few hours, it is more expensive and unsatisfactorily sensitive relative to the culture method. We have developed a method to rapidly detect Salmonella enterica by using a compact disc (CD)-shaped device that can reduce reagent consumption in conventional PCR. The detection method, which combines culture and PCR, is more rapid than the conventional culture method and is more sensitive and cheaper than PCR. In this study, we also examined a sample preparation method that involved collecting bacterial cells from food. The bacteria collected from chicken meat spiked with S. enterica were mixed with PCR reagents, and PCR was performed on the device. At a low concentration of S. enterica, the collected S. enterica was cultured before PCR for sensitive detection. After cultivation for 4 h, S. enterica at 1.7 × 104 colony-forming units (CFUs)·g-1 was detected within 8 h, which included the time needed for sample preparation and detection. Furthermore, the detection of 30 CFUs·g-1 of S. enterica was possible within 12 h including 8 h for cultivation.

Clinical Significance of Neoadjuvant Combined Androgen Blockade for More Than Six Months in Patients with Localized Prostate Cancer Treated with Prostate Brachytherapy.

INTRODUCTION: The aim of this study is to clarify the clinical significance of neoadjuvant combined androgen blockade (CAB) for ≥ 6 months in patients with localized prostate cancer. PATIENTS AND METHODS: A total of 431 patients with localized prostate cancer who underwent prostate brachytherapy (BT) with or without neoadjuvant CAB for ≥ 6 months with mean follow-up time of 64.6 months (range 24-108 months) were evaluated retrospectively. Of those 431, 232 patients received BT in combination with neoadjuvant CAB for ≥ 6 months. Biochemical recurrence-free rates (BRFRs) in 364 patients with at least 3 years of follow-up were evaluated by log-rank test. RESULTS: BRFR in patients with low-, intermediate- and high-risk prostate cancer were 98.1, 94.2 and 89.1%, respectively. In patients with intermediate-risk prostate cancer only, neoadjuvant CAB was significantly associated with BRFR (p = 0.0468). Especially in patients with intermediate-risk prostate cancer with radiation dose received by 90% of the prostate (D90) < 180 Gy, neoadjuvant CAB exerted a favorable impact on BRFR (p = 0.0429). On multivariate analyses, neoadjuvant CAB and D90 were independent predictors of BRFR (p = 0.0061 and p < 0.0001, respectively). CONCLUSIONS: Neoadjuvant CAB for ≥ 6 months has a favorable impact on BRFR in patients with intermediate-risk prostate cancer, particularly in patients with relatively low radiation doses of D90.

Rapid and highly sensitive detection by a real-time polymerase chain reaction using a chip coated with its reagents.

On-site detection by flow-through polymerase chain reaction (PCR) microfluidic systems for rapid and highly sensitive analysis, are significantly desired for bioanalytical and medical research. The conventional continuous-flow PCR chips realized rapid detection, but their sensitivity was very low (10(6) to 10(8) copies µL(-1)). We improved this drawback by coating the chip with a PCR reagents mixture, and succeed to obtain a rapid and highly sensitive detection by using a segment-flow PCR system. In the present work, we developed a portable segment-flow PCR system for practical use. PCR was performed for the uid A gene in E. coli. By real-time segment-flow PCR using coated chips, we realized rapid detection in 8 min and a high sensitivity of 4 cells µL(-1). The sensitivity by the segment-flow PCR chip was the same as that of a conventional thermal cycler. Moreover, the detection speed of our segment-flow PCR chip was 15-times as rapid as that of the conventional thermal cycler.

Changes of tumor and normal structures of the neck during radiation therapy for head and neck cancer requires adaptive strategy.

The treatment period over which radiation therapy is administered extends over several weeks. Since tumor shrinkage in response to radiation therapy and weight loss due to radiation-induced mucositis may impact on the dose distribution in both target and organ at risk in patients with head and neck cancer, the anatomical changes of tumor and neck volumes during this period should be taken into consideration. We investigated the anatomical changes that occurred in the target and normal structure of the neck during radiation therapy for pharyngeal cancer, and evaluated the necessity of an adaptive strategy. Ten patients with pharyngeal cancer who underwent radical chemoradiation therapy using 3-dimensional conformal radiation therapy RT (66-70 Gy in 33-35 fractions) between April 2009 and September 2010 were enrolled in the study. Patients underwent CT scans every week during their course of treatment. We analyzed the CT data in the radiation treatment planning system and measured changes of tumor, organ at risk, and neck volume. Gross tumor volume (GTV) was rapidly reduced by 28% of the original volume on average in the first 3 weeks. The right and left submandibular glands volume decreased to 70% and 63% of their initial volumes on average, respectively. The volume of the neck in the radiation fields decreased to 89% of its initial volume on average by the sixth week mainly caused by body weight loss due to acute radiation morbidity. Considerable anatomical change in the radiation filed that will affect dose distribution of the target and organ at risk was observed during radiation therapy for head and neck cancer.

Detection of expressed gene in isolated single cells in microchambers by a novel hot cell-direct RT-PCR method.

In order to be able to detect the expression of a gene in individual cells, the ability to isolate and lyse a single cell and to perform reverse transcription polymerase chain reaction (RT-PCR) in one device is important. As is common, when performing cell lysis and RT-PCR in the same reaction chamber, it is necessary to add the reagent for RT-PCR after cell lysis. In this study, we propose an original formula for cell lysis and RT-PCR in the same reaction chamber without the addition of reagent by only a heat process, which we termed hot cell-direct RT-PCR. Hot cell-direct RT-PCR was enabled by using Tth DNA polymerase, which is a thermostable polymerase and has high reverse transcription activity in the presence of manganese ions. Direct detection of RT-PCR products was performed by detecting fluorescence with the use of a double-dye fluorescent probe. We attempted to detect the mRNA of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene in isolated Jurkat cells on a microfluidic device, which we had already developed for single cell isolation. After cell isolation and successive hot cell-direct RT-PCR on the device, fluorescent signals from RT-PCR products for a single cell were detected and differentiated from the chamber containing no cells. A highly positive linear relationship (r = 0.9933) was observed between the number of chambers containing cell(s) and those containing RT-PCR products from 10 to 400 cells µL(-1). Thus it was possible to use the novel hot cell-direct RT-PCR method to detect the expressed gene in isolated cells.

Use of a novel microfluidic disk in the analysis of single-cell viability and the application to Jurkat cells.

Jurkat cells were trapped in the microchambers of a novel disk-shaped cell separation device and stained with Cellstain. Approximately 90% of the cells were living. Single cells were isolated with a branching microchannel after rotation at 4500rpm for 30s, demonstrating that a living single cell could be trapped in the microchambers.

Compact disk (CD)-shaped device for single cell isolation and PCR of a specific gene in the isolated cell.

For immediate discrimination among isolated cells we propose a novel device and technique for isolation of cells and sequential detection of specific gene(s) within them by polymerase chain reaction (PCR). In this study, we isolated Salmonella enterica cells and detected the Salmonella-specific invA gene from isolated cells by PCR on a compact disk (CD)-shaped device. This device enabled liquid flow by centrifugal force without a micro pump, and was fabricated from silicon wafer and glass to avoid evaporation of a small amount of reagent. One device has 24 microchannels, and 313 microchambers integrated on each microchannel. One microliter of PCR mixture containing cells was separated into microchambers on the device at 5000 rpm for 30 s. Each microchamber contained approximately 1.5 nL PCR mixture. A Poisson distribution of S. enterica cells was observed for different densities of cell suspension. At 200 cells µL(-1) of S. enterica or less, isolated single cells could be determined on the device by amplification of DNA of the invA gene; at 400 cells µL(-1), chambers containing no, one, two, or three cells could be determined on the device. Selective detection of S. enterica was achieved by PCR from a mixture of S. enterica and Escherichia coli on the CD-shaped device.

High-dose-rate brachytherapy for patients with maxillary gingival carcinoma using a novel customized intraoral mold technique.

OBJECTIVE: The purpose of this study was to introduce a novel customized intraoral mold treatment for maxillary gingival carcinoma (UGC). STUDY DESIGN: Two patients with UGC were treated as salvage therapy using this technique. The mold was designed to keep normal soft tissues adjacent to the tumor away from the radioactive source as much as possible, and it was shielded by lead. The radiation dose on the buccal mucosa and tongue was measured at the inner and outer surfaces of the intraoral mold before starting high-dose-rate brachytherapy by the remote afterloading system, and was reduced to almost one tenth. RESULTS: The patient had no recurrence and no severe adverse effects on the normal soft tissue adjacent to the tumor until the end of the follow-up period. CONCLUSION: High-dose-rate brachytherapy using the novel customized intraoral mold might be a treatment option of not only salvage therapy, but definitive therapy of UGC.