Value of gene polymorphisms as markers of 5-FU therapy response in stage III colon carcinoma: A pilot study

Purpose: The role of pharmacogenetics in chemotherapy response in colon carcinoma is controversial. We studied the value of known SNPs in genes involved in 5-FU metabolism as biomarkers of chemotherapy response in patients with stage III colon carcinoma. Methods: DNA was isolated from normal colonic tissue of 60 patients with stage III colon carcinoma treated adjuvantly with 5-FU combined with leucovorin. The tested SNPs were validated SNPs on the OPRT, TYMS and DPYD genes and a synonymous SNP on the TYMP gene. Real-time PCR, sequencing and RFLP were used for genotyping. Results: None of the studied genotypes was associated with any of the tumor or patient characteristics. Moreover, none of the genotypes studied had effect on patient survival. Conclusion: In conclusion, the tested SNPs are not biomarkers of chemotherapy response in our stage III colon cancer patients group

Rapid genotyping of human papillomavirus by post-PCR array-based hybridization techniques

Kinetic hybridization measurements on a microarray are expected to become a valuable tool for genotyping applications. A method has been developed that enables kinetic hybridization measurements of PCR products on a low-density microarray. This is accomplished by pumping a solution containing PCR products up and down through a porous microarray substrate. After every pumping cycle, the fluorescently labeled PCR products hybridized to capture probes immobilized on the solid surface of the porous microarray substrate are measured. By this method, both binding curves and high-resolution melting curves are obtained instead of the single endpoint hybridization intensities as with commonly used post-PCR array-based hybridization techniques. We used 20 subtypes of the human papillomavirus (HPV) as a model system to test our detection method and blindly analyzed 216 clinical samples. We compared our microarray flowthrough method with a reference method, PCR followed by a reverse line blot (RLB). Real-time hybridization measurements followed by high-resolution melting curves of low concentrations of fluorescently labeled HPV targets on a microarray were successfully carried out without any additional chemical signal amplification. The results of our new method were in good agreement (93%, with a kappa coefficient of κ = 0.88 [95% CI, 0.81 to 0.94]) with the RLB results. All discrepant samples were analyzed by a third method, enzyme immunoassay (EIA). Furthermore, in a number of cases, we were able to identify false-positive samples by making use of the information contained in the kinetic binding and melting curves. This clearly demonstrates the added value of the use of kinetic measurements and high-resolution melting curves, especially for highly homologous targets

Biomarkers and Molecular Analysis to Improve Bloodstream Infection Diagnostics in an Emergency Care Unit

Molecular pathogen detection from blood is still expensive and the exact clinical value remains to be determined. The use of biomarkers may assist in preselecting patients for immediate molecular testing besides blood culture. In this study, 140 patients with ≥ 2 SIRS criteria and clinical signs of infection presenting at the emergency department of our hospital were included. C-reactive protein (CRP), neutrophil-lymphocyte count ratio (NLCR), procalcitonin (PCT) and soluble urokinase plasminogen activator receptor (suPAR) levels were determined. One ml EDTA blood was obtained and selective pathogen DNA isolation was performed with MolYsis (Molzym). DNA samples were analysed for the presence of pathogens, using both the MagicPlex Sepsis Test (Seegene) and SepsiTest (Molzym), and results were compared to blood cultures. Fifteen patients had to be excluded from the study, leaving 125 patients for further analysis. Of the 125 patient samples analysed, 27 presented with positive blood cultures of which 7 were considered to be contaminants. suPAR, PCT, and NLCR values were significantly higher in patients with positive blood cultures compared to patients without (p < 0.001). Receiver operating characteristic curves of the 4 biomarkers for differentiating bacteremia from non-bacteremia showed the highest area under the curve (AUC) for PCT (0.806 (95% confidence interval 0.699–0.913)). NLCR, suPAR and CRP resulted in an AUC of 0.770, 0.793, and 0.485, respectively. When compared to blood cultures, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for SepsiTest and MagicPlex Sepsis Test were 11%, 96%, 43%, 80%, and 37%, 77%, 30%, 82%, respectively. In conclusion, both molecular assays perform poorly when one ml whole blood is used from emergency care unit patients. NLCR is a cheap, fast, easy to determine, and rapidly available biomarker, and therefore seems most promising in differentiating BSI from non-BSI patients for subsequent pathogen identification using molecular diagnostics

Historia natural de la infección por el virus del papiloma humano: un estudio de cinco años de seguimiento

&lt;p class="MsoNormal"&gt;&lt;span style="font-size: 10pt; font-family: Arial"&gt;Introducción: El virus del papiloma humano es el principal factor de riesgo asociado con el desarrollo de cáncer cervical, uno de los cánceres más comunes en mujeres colombianas. En Colombia, donde las tasas de incidencia de cáncer cervical son altas, se tiene poca información acerca de la prevalencia de la infección y no se tienen datos de seguimiento sobre la persistencia o eliminación de la infección y los factores de riesgo asociados. En este reporte se presentan los resultados de la prevalencia de la infección por HPV y los factores de riesgo asociados en mujeres que tuvieron una citología normal al comienzo del estudio así como el seguimiento de 227 mujeres positivas para la infección por HPV durante un periodo de cinco años, en la ciudad de Bogotá, Colombia.&lt;/span&gt;&lt;/p&gt; &lt;p class="MsoNormal"&gt;&lt;span style="font-size: 10pt; font-family: Arial"&gt;Materiales y Métodos: 1859 mujeres con citología normal firmaron un consentimiento escrito y respondieron a un cuestionario estructurado. La presencia de HPV fue analizada mediante PCR con el uso de los iniciadores GP5+/ GP6+, que amplifican para un fragmento de 140 pb de la región L1/HPV (1) y tipificadas por medio del ensayo de EIA para la identificación de 37 diferentes tipos de HPV (2).&lt;/span&gt;&lt;/p&gt; &lt;p class="MsoNormal"&gt;&lt;span style="font-size: 10pt; font-family: Arial"&gt;Se calcularon riesgos relativos para eliminación de la infección por HPV mediante el uso de curvas de supervivencia. &lt;/span&gt;&lt;/p&gt; &lt;p class="MsoNormal"&gt;&lt;span style="font-size: 10pt; font-family: Arial"&gt;Resultados: La prevalencia de la infección fue de 14.8%; 9% de las mujeres fueron infectadas por HPV de alto riesgo, 3.1% por HPV de bajo riesgo, 2.3% por ambos tipos virales y 0.4% por HPV indeterminados. Se identificaron 32 diferentes tipos de HPV, siendo el HPV16, 58, 56, 81 y 18, los tipos mas comunes. La prevalencia de la infección por HPVs de alto riesgo entre mujeres jóvenes (menores de 20 años) fue de 26.1%, en mujeres con edades entre 45 y 54 años fue de 2.3% y en mujeres mayores de 55 años o mas fue de 13.2%. Para HPV de bajo riesgo, el mayor pico de prevalencia se observó en mujeres mayores de 55 años. Comparadas con mujeres en edades entre 35-44 años, las mujeres menores de 20 años tuvieron un mayor riesgo (10 veces) de tener infecciones múltiples. La edad, el número de compañeros sexuales regulares y el uso de anticonceptivos orales fueron los principales factores de riesgos asociados con la infección por HPV. En mujeres menores de 25 años, el alto nivel educacional y el tener compañeros sexuales casuales fueron factores de riesgo asociados con la infección. Durante el seguimiento de las las infecciones por HPV, 16 tuvieron una tasa de eliminación significativamente mas lenta que las infecciones por HPV de bajo riesgo (RR = 0.47, 95% IC: 0.32-0.72), tipos relacionados a HPV 16 (tipos 31, 33, 35, 52, 58) tuvieron tasas de eliminación intermedias (RR = 0.62, 95% IC: 0.47-0.94), los demás tipos de alto riesgo no mostraron diferencias en la tasa de eliminación cuando se compararon con HPVs de bajo riesgo. Infecciones singulares y múltiples tuvieron tasas de eliminación similares. Tampoco se observó una evidencia clara de asociación entre carga viral y eliminación de la infección. En mujeres con paridades se observó una tasa de eliminación de la infección mas lenta que en mujeres nulíparas (RR = 0.64, 95% IC: 0.47-0.89) y en mujeres que han usado alguna vez anticonceptivos, orales se observó una tasa de eliminación de la infección mas rápida que en mujeres que no han usado anticonceptivos orales (RR = 1.38, 95% IC: 1.07-1.77).&lt;/span&gt;&lt;/p&gt; &lt;p class="MsoNormal"&gt;&lt;span style="font-size: 10pt; font-family: Arial"&gt;Conclusión: Hay una amplia diversidad de infecciones por HPV en la cohorte analizada, siendo los HPV de alto riesgo los tipos más comunes. En esta población múltiples compañeros sexuales y entre mujeres jóvenes un alto nivel de educación y compañeros sexuales casuales parecen determinar el riesgo de infección. Las infecciones por HPV16 son eliminadas en un periodo mas largo que otras infecciones por HPVs de alto y bajo riesgo.&lt;/span&gt;&lt;/p&gt; &lt;p class="MsoNormal"&gt;&lt;span style="font-size: 10pt; font-family: Arial"&gt; &lt;/span&gt;&lt;/p&gt; &lt;p class="MsoNormal"&gt;&lt;span style="font-size: 10.5pt; font-family: Arial"&gt;REFERENCIAS&lt;/span&gt;&lt;/p&gt; &lt;p class="MsoNormal"&gt;&lt;span style="font-size: 9pt; font-family: Arial"&gt;1. DE RODA HUSMAN AM, WALBOOMERS JMM, VAN DER BRULE AJC, MEIJER CJLM AND SNIJDERS PJF. The use general primers GP5 and GP6 elongated at their 3' ends with adjacent highly conserved sequences improves human papilomavirus detection by PCR. J Gen Virol 1995; 76:1057-62.&lt;/span&gt;&lt;/p&gt; &lt;p class="MsoNormal"&gt;&lt;span style="font-size: 9pt; font-family: Arial"&gt;2. JACOBS MV, WALBOOMERS JMM, SNIJDERS PJF, VOORHORST FJ, DAALMEIJER NF AND MEIJER CJLM. Age-related distribution patterns of 37 mucosotropic HPV types in women with citologically normal cervical smears: Decreased high risk/low risk ratio at older age. Int J Cancer 2000; 87(2): 221-27.&lt;/span&gt;&lt;/p&gt

SNaPshot and StripAssay as Valuable Alternatives to Direct Sequencing for KRAS Mutation Detection in Colon Cancer Routine Diagnostics

Although direct sequencing is the gold standard for KRAS mutation detection in routine diagnostics, it remains laborious, time consuming, and not very sensitive. Our objective was to evaluate SNaPshot and the KRAS StripAssay as alternatives to sequencing for KRAS mutation detection in daily practice. KRAS exon 2–specific PCR followed by sequencing or by a SNaPshot reaction was performed. For the StripAssay, a mutant-enriched PCR was followed by hybridization to KRAS-specific probes bound to a nitrocellulose strip. To test sensitivities, dilution series of mutated DNA in wild-type DNA were made. Additionally, direct sequencing and SNaPshot were evaluated in 296 colon cancer samples. Detection limits of direct sequencing, SNaPshot, and StripAssay were 20%, 10%, and 1% tumor cells, respectively. Direct sequencing and SNaPshot can detect all 12 mutations in KRAS codons 12 and 13, whereas the StripAssay detects 10 of the most frequent ones. Workload and time to results are comparable for SNaPshot and direct sequencing. SNaPshot is flexible and easy to multiplex. The StripAssay is less time consuming for daily laboratory practice. SNaPshot is more flexible and slightly more sensitive than direct sequencing. The clinical evaluation showed comparable performances between direct sequencing and SNaPshot. The StripAssay is rapid and an extremely sensitive assay that could be considered when few tumor cells are available. However, found mutants should be confirmed to avoid risk of false positives