Diagnostic microRNA markers to screen for sporadic human colon cancer in stool: I. Proof of principle.

To present proof-of-principle application for employing micro(mi)RNAs as diagnostic markers for colon cancer, we carried out global microarray expression studies on stool samples obtained from fifteen individuals (three controls, and three each with TNM stage 0-1, stage 2, stage 3, and stage 4 colon cancer), using Affymetrix GeneChip miRNA 3.0 Array, to select for a panel of miRNA genes for subsequent focused semi-quantitative polymerase chain reaction (PCR) analysis studies. Microarray results showed 202 preferentially expressed miRNA genes that were either increased (141 miRNAs), or reduced (61 miRNAs) in expression. We then conducted a stem-loop reverse transcriptase (RT)-TaqMan® minor groove binding (MGB) probes, followed by a modified qPCR expression study on 20 selected miRNAs. Twelve of the miRNAs exhibited increased and 8 decreased expression in stool from 60 individuals (20 controls, 20 with tumor-lymph node-metastatic (TNM) stage 0-1, 10 with stage 2, five with stage 3, and 5 with stage 4 colon cancer) to quantitatively monitor miRNA changes at various TNM stages of colon cancer progression. We also used laser-capture microdissection (LCM) of colon mucosal epithelial tissue samples (three control samples, and three samples from each of the four stages of colon cancer, for a total of 15 samples) to find concordance or lack thereof with stool findings. The reference housekeeping pseudogene-free ribosomal gene (18S rRNA), which shows little variation in expression, was employed as a normalization standard for relative PCR quantification. Results of the PCR analyses confirmed that twelve miRNAs (miR-7, miR-17, miR-20a, miR-21, miR-92a, miR-96, miR-106a, miR-134, miR-183, miR-196a, miR-199a-3p and miR214) had an increased expression in the stool of patients with colon cancer, and that later TNM carcinoma stages exhibited a more pronounced expression than did adenomas. On the other hand, eight miRNAs (miR-9, miR-29b, miR-127-5p, miR-138, miR-143, miR-146a, miR-222 and miR-938) had decreased expression in the stool of patients with colon cancer, which was also more pronounced from early to later TNM stages. Results from colon mucosal tissues were similar to those from stool samples, although with more apparent changes in expression. Cytological studies on purified stool colonocytes that employed Giemsa staining showed 80% sensitivity for detecting tumor cells in stool smears. The performance characteristics of the test confirmed that stool is a medium well-suited for colon cancer screening, and that the quantitative changes in the expression of few mature miRNA molecules in stool associated with colon cancer progression provided for more sensitive and specific non-invasive diagnostic markers than tests currently available on the market. Thus, a larger prospective and properly randomized validation study of control individuals and patients exhibiting various stages of colon cancer progression (TNM stages 0-IV) is now needed in order to standardize test conditions, and provide a means for determining the true sensitivity and specificity of a miRNA screening approach in stool for the non-invasive detection of colon cancer, particularly at an early stage (0-I). Eventually, we will develop a chip to enhance molecular screening for colon cancer, as has been accomplished for the detection of genetically-modified organisms (GMOs) in foods.

Total error shift patterns for daily CT on rails image-guided radiotherapy to the prostate bed.

BACKGROUND: To evaluate the daily total error shift patterns on post-prostatectomy patients undergoing image guided radiotherapy (IGRT) with a diagnostic quality computer tomography (CT) on rails system. METHODS: A total of 17 consecutive post-prostatectomy patients receiving adjuvant or salvage IMRT using CT-on-rails IGRT were analyzed. The prostate bed's daily total error shifts were evaluated for a total of 661 CT scans. RESULTS: In the right-left, cranial-caudal, and posterior-anterior directions, 11.5%, 9.2%, and 6.5% of the 661 scans required no position adjustments; 75.3%, 66.1%, and 56.8% required a shift of 1 - 5 mm; 11.5%, 20.9%, and 31.2% required a shift of 6 - 10 mm; and 1.7%, 3.8%, and 5.5% required a shift of more than 10 mm, respectively. There was evidence of correlation between the x and y, x and z, and y and z axes in 3, 3, and 3 of 17 patients, respectively. Univariate (ANOVA) analysis showed that the total error pattern was random in the x, y, and z axis for 10, 5, and 2 of 17 patients, respectively, and systematic for the rest. Multivariate (MANOVA) analysis showed that the (x,y), (x,z), (y,z), and (x, y, z) total error pattern was random in 5, 1, 1, and 1 of 17 patients, respectively, and systematic for the rest. CONCLUSIONS: The overall daily total error shift pattern for these 17 patients simulated with an empty bladder, and treated with CT on rails IGRT was predominantly systematic. Despite this, the temporal vector trends showed complex behaviors and unpredictable changes in magnitude and direction. These findings highlight the importance of using daily IGRT in post-prostatectomy patients.

Surface plasmon resonance (SPR) spectrometry as a tool to analyze nucleic acid-protein interactions in crude cellular extracts.

This study presents proof-of-principle application showing that label-free affinity enrichment surface plasmon resonance (SPR) biosensor binding is able to semiquantitatively detect molecular DNA-protein interactions in crude cellular extracts in a real-time ligand fishing analysis study. Crude cell extracts obtained from a confluent HT-28 human adenocarcinoma cell line, synchronized to the G(0)/G(1) phase of the cell cycle, were extracted in a chaotropic medium and cryopreserved in liquid nitrogen. Various immunoprecipitation antibodies were used against defective human excision and mismatch repair genes, hDDB2 and hMSH2, respectively, which theoretically allow for protein binding to DNA ligands in their native conformation. A set of biotinylated DNA target sequence heteroduplexes were also utilized for binding hDDB2 and hMSH2, prepared by heating a biotinylated oligonucleotide strand with an equimolar amount of the complementary strand to form a DNA duplex for hMSH2; a UV-irradiated duplex was employed for hDDB2 instead of an irradiated single-strand DNA to enhance binding. SDS was used to regenerate heteroduplex-modified chips that were used in a BIAcore 2000 SPR instrument at 25°C. Results showed that hMSH2 does not bind preferentially to the heteroduplex-complementary pair. In contrast, hDDB2 was found to bind preferentially to the UV-irradiated version of the heteroduplex-complementary pair. It is concluded that the choice of antibodies with appropriate epitopes is crucial to the success of these SPR binding studies because of enhanced specificity.

Study of a spherical phantom for Gamma knife dosimetry.

Four 16 cm diameter spherical phantoms were modeled in this study: a homogenous water phantom, and three water phantoms with 1 cm thick shell each made of different materials (PMMA, Plastic WaterTM and polystyrene). The PENELOPE Monte Carlo code was utilized to simulate photon beams from the Leksell Gamma Knife (LGK) unit and to determine absorbed dose to water (Dw) from a single 18 mm beam delivered to each phantom. A score spherical volume of 0.007 cm3 was used to simulate the dimensions of the sensitive volume of the Exradin A-16 ionization chamber, in the center of the phantom. In conclusion, the PMMA shell filled with water required a small correction for the determination of the absorbed dose, while remaining within the statistical uncertainty of the calculations (+/- 0.71). Plastic WaterTM and polystyrene shells can be used without correction. There is a potential advantage to measuring the 4 mm helmet output using these spherical water phantoms.

Identification and management of persistently active brachytherapy seed implants.

OBJECTIVE: The purpose of this article is to review the history of permanently implanted brachytherapy sources and to establish methods of identifying radon sources and discussing appropriate management. CONCLUSION: There are likely thousands of people bearing radon seeds that continue to emit radiation decades after implantation. They can be identified by clinical history and emission of characteristic x-rays. Surgical removal of these sources is rarely warranted.

Differences in mRNA and microRNA microarray expression profiles in human colon adenocarcinoma HT-29 cells treated with either Intensity-modulated Radiation Therapy (IMRT), or Conventional Radiation Therapy (RT).

We carried out this in vitro molecular study to investigate the effect of two clinical X-irradiation modalities (a two-dimensional external beam radiotherapy referred to in this article as conventional RT, and a three dimensional conformal intensity-modulated radiation therapy (IMRT) on a colon adenocarcinoma HT-29 cell line. Cells were synchronized by serum deprivation 48 h before irradiation so that >90% of them were in the G(0)/G(1) phase of the cell cycle. Cells were allowed to recover 3 h after irradiation before total RNA extraction. Two types of arrays, namely Affymetrix Human HG U133A 2.0 oligonucleotide microarrays and Ambion mirVana bioarrays, were employed to study mRNA and microRNA expressions, respectively. Three flasks were used per irradiation dose, and an additional three unirradiated flasks served as control. Microarray data were validated by reverse transcriptase quantitative polymerase chain reaction, and proteins of some expressed genes were determined by Western blots. Results showed the existence of differences in expression profiles between the two irradiation modalities. IMRT appeared to influence expression of some DNA repair genes, whereas in conventional RT, some DNA repair and cell cycle-related genes that initially seemed to be preferentially expressed dwindled to normal levels. Earlier in vitro experiments using cell survival to study sublethal damage repair support our conclusions. Bioinformatic investigation revealed a correlation of gene expression with derepression effects of microRNA molecules. We have presented opinions as to how microRNAs might influence gene expression during radiation-induced stress and have suggested future avenues for research.

Image-guided radiation therapy: current and future directions.

Since its discovery, ionizing radiation has been a cornerstone of cancer treatment. In step with technological advances, radiation therapy has strived to increase its therapeutic ratio. With the advent of 3D and cross-sectional imaging, and the ability to modulate the radiation beam, the current age of radiation oncology was initiated, promising better tumor control rates with fewer side effects. However, these ever more precise and conformal treatments have also revealed the importance of accounting for organ and tumor motion. Efforts to understand and compensate for the uncertainties caused by movement are required to ensure accurate conformal radiation therapy. This review will explore the current and future directions of image-guided radiation therapy, whose goal is to increase the accuracy of radiotherapy.

An ultrasonic device for source to skin surface distance measurement in patient setup.

PURPOSE: To develop an ultrasound-based source to skin surface distance (SSD) measurement technique and device for patient setup and test its feasibility and accuracy. METHODS AND MATERIALS: The ultrasonic SSD measurement device (USD) prototype consists of two main parts: a probe plate with an ultrasonic transducer in the center and a control unit that displays the SSD in millimeters. The probe plate can be slid into the block tray accessory slot of any treatment machine at the time of the SSD measurement. The probe plate contains an ultrasonic transducer as both the source and the detector for measuring the distance between the transducer and the target surfaces on the basis of an echo-detecting technique. The device was calibrated by a mechanical ruler with an accuracy of 0.01 mm and corrected by an offset of 601.7 mm, which is the distance from the radiation source to the ultrasonic transducer surface for the Siemens Primus linear accelerator (Linac). The ultrasound device provided digital readout with an accuracy of +/-0.1 mm for a flat surface after calibration. The SSD measurement experiments were done with the USD, an optical distance indicator (ODI), and an AKTINA 53-104 Mechanical Front Pointer (FP) on a Siemens Primus Linac with a full-sized female phantom. Ten measurements were carried out at each gantry angle of 0 degrees , 52 degrees , 85 degrees , 90 degrees , and 227 degrees for anatomic locations of head, thorax, breast, and pelvis, to obtain the average values and standard deviations. RESULTS: The comparison study with the ODI and FP showed that the USD had an accuracy of less than +/-1.0 mm and that USD measurements had the minimum standard deviations among the three methods; therefore, USD gave more consistent and accurate readouts for SSD measurement. When considering the FP as a reference, the USD yields smaller deviations than the ODI for all measured locations (less than +/-2 mm). The variation of USD digital readout with a room temperature change of +/-2 degrees C is +/-0.1 mm, which is sufficiently accurate for SSD measurement. CONCLUSIONS: The USD method has the following advantages. First, it decreases patient setup time by avoiding problems related to the blocking of the device by the patient or by the immobilization device. Second, it is more accurate than the other two methods currently used, as the test data show. Last, the digital readout eliminates the possibility of human reading error associated with the visual scales.

Verificação do fator de calibração e indicador da qualidade do feixe de aceleradores lineares / Verification of dose output and beam quality indicator in linear accelerators

Um programa de garantia da qualidade é um pré-requisito obrigatório para a exatidão necessária em radioterapia. Este trabalho relata parte dos testes de rotina mensal do controle da qualidade dos aceleradores lineares do Instituto Nacional do Câncer, relativos à calibração dos feixes de fótons e elétrons, no período de dois anos. Os resultados foram comparados com as recomendações do protocolo AAPM TG-40. Na análise do fator de calibração para o feixe de fótons foi encontrado um desvio máximo de 12 por cento; para o feixe de elétrons o maior desvio encontrado foi 10 por cento. A flutuação observada no indicador da qualidade do feixe para os feixes de elétrons foi maior que para os feixes de fótons. Os resultados confirmam a importância de um programa de garantia da qualidade em um serviço de radioterapia, permitindo correções rápidas da dose administrada ao paciente.(AU)#S#a

Avaliaçäo de proteçäo ocular em feixes de elétrons / Evaluation of eye shield for electron beams

Nos tratamentos por radiaçäo, nos quais a regiäo ocular é exposta ao campo de tratamento, uma proteçäo é colocada entre a pálpebra e o globo ocular para proteger as estruturas oculares mais sensíveis à radiaçäo. Isto é importante, pois uma dose de 500 cGy na córnea pode provocar catarata. A efetividade de uma proteçäo ocular construida em chumbo e uma outra proteçäo comercial (Ace Medical Supply Co., New York) foi avaliada para feixes de elétrons de 5,4 MeV e 3,7 MeV. Na avaliaçäo do efeito de concavidade, verificou-se que o formato côncavo desse tipo de proteçäo permite que o dobro de radiaçäo seja transmitida, comparada a uma proteçäo plana. A proteçäo comercial testada apresentou proteçäo suficiente para o feixe de 3,7 MeV/ entretanto, para o feixe de 5,4 MeV a dose na interface proteçäo-material absorvedor foi 56 por cento da dose na profundidade de dose máxima, dmax, sem a proteçäo. A espessura encontrada para uma dose transmitida de aproximadamente 5 por cento da dose em dmax`, para uma proteçäo desse tipo, para este último feixe, foi de 3mm em chumbo. Um aumento significativo da dose na interface externa da proteçäo ocorre devido ao efeito de retroespalhamento dos elétrons. Para que este efeito seja reduzido a um nível de dose próximo à dose em dmax, espessura mínima de 3mm de poliestireno é necessária para ambos os feixes testados

Medidas dos fatores de conversäo H*(d)/Ka pra feixes de raios-X diagnóstico / Measure of H*(d)Ka conversion for Diagnostic X-rays fields

Este trabalho apresenta os resultados da determinação experimental dos coeficientes de conversão da grandeza de calibração kerma no ar para a nova grandeza da ICRU de monitoração de área, H*(d). As medidas foram realizadas nas profundidades 10, 50 e 60 mm de uma esfera de PMMA de 30 cm de diâmetro em feixes de raios-X de radiodiagnóstico.