Clinical and pathological features of BRCA1 associated carcinomas in a hospital-based sample of Dutch breast cancer patients

Thus far, studies investigating the differences in tumour characteristics between breast cancer in BRCA1-carriers and other patients, have focused on highly selected groups of patients, potentially limiting the conclusions that can be drawn. Previously, we had identified 10 patients with BRCA1 germline mutations in a hospital-based series of 642 breast cancer patients not selected for age or family history. The aim of this analysis is to investigate the clinical and pathological features of these BRCA1 associated carcinomas as compared to other breast cancers in this representative sample. Tumours from patients with BRCA1 germline mutations (n= 10) were compared to an age-matched sample of other patients (n= 50) from the same cohort. The following characteristics were considered: axillary nodal status and tumour size, histologic parameters (tumour type, histologic grade, mitotic rate, tubule formation, nuclear grade, CIS and lymphangio invasion) and expression of several proteins (oestrogen and progesterone receptors, cyclin D1, p53, HER2/neu, E-cadherin). In BRCA1associated tumours receptors for oestrogen and progesterone were expressed less frequently (respectively P= 0.001 and P= 0.002) than in controls, which is in line with findings from other studies. Other differences were also in accordance with findings from other studies, although not statistically significant. We conclude that the features of BRCA1 associated tumours detected in a hospital-based series of breast cancer patients not selected for family history of age at diagnosis are similar to tumours in cases selected for family history or age at diagnosis. © 2001 Cancer Research Campaign  http://www.bjcancer.co

Noninvasive multianalyte diagnostic assay for monitoring bladder cancer recurrence

Neal D Shore,1 Cecilia A Fernandez,2 Anthony P Shuber21Carolina Urologic Research Center/Atlantic Urology Clinics, Myrtle Beach, SC, 2Predictive Biosciences Inc, Lexington, MA, USABackground: The purpose of this study was to establish the clinical performance of a urine-based assay, called a multianalyte diagnostic readout, in monitoring for bladder cancer recurrence.Methods: This was a prospective, multicenter, single assessment observational study. The multianalyte diagnostic readout uses a combination of one protein and three DNA biomarkers. Urine samples from 733 patients undergoing monitoring for bladder cancer recurrence were analyzed for matrix metalloproteinase-2 levels, the presence of mutant FGFR3 DNA, and hypermethylation of the NID2 and VIM genes. The probability of a patient having (positive predictive value) or not having (negative predictive value) recurrent bladder cancer was determined by FGFR3 alone or all four biomarkers combined, respectively.Results: Cystoscopy/biopsy diagnosed 63 patients with bladder cancer recurrence at the time of study assessment. The four-biomarker assay identified 237 patients as having a low probability of disease recurrence, 231 of whom were determined by cystoscopy as not having recurrent cancer, resulting in a negative predictive value of 97.5% at 90.5% sensitivity. The FGFR3 assay identified 49 patients with FGFR3 mutations, 19 of whom were confirmed by biopsy as having cancer, resulting in a positive predictive value of 38.8%, with 95.5% specificity.Conclusion: The urine-based multianalyte diagnostic readout assay was able to delineate the patient population into those highly likely to have bladder cancer recurrence, those unlikely to have recurrent disease, and those with an average risk for bladder cancer recurrence.Keywords: FGFR3, matrix metalloproteinase-2, NID2, VIM, recurrent bladder cance

Detection of low frequency FGFR3 mutations in the urine of bladder cancer patients using next-generation deep sequencing

John M Millholland, Shuqiang Li, Cecilia A Fernandez, Anthony P ShuberPredictive Biosciences Inc, Lexington, MA, USAAbstract: Biological fluid-based noninvasive biomarker assays for monitoring and diagnosing disease are clinically powerful. A major technical hurdle for developing these assays is the requirement of high analytical sensitivity so that biomarkers present at very low levels can be consistently detected. In the case of biological fluid-based cancer diagnostic assays, sensitivities similar to those of tissue-based assays are difficult to achieve with DNA markers due to the high abundance of normal DNA background present in the sample. Here we describe a new urine-based assay that uses ultradeep sequencing technology to detect single mutant molecules of fibroblast growth factor receptor 3 (FGFR3) DNA that are indicative of bladder cancer. Detection of FGFR3 mutations in urine would provide clinicians with a noninvasive means of diagnosing early-stage bladder cancer. The single-molecule assay detects FGFR3 mutant DNA when present at as low as 0.02% of total urine DNA and results in 91% concordance with the frequency that FGFR3 mutations are detected in bladder cancer tumors, significantly improving diagnostic performance. To our knowledge, this is the first practical application of next-generation sequencing technology for noninvasive cancer diagnostics.Keywords: FGFR3, mutation, urine, single molecule, sequencing, bladder cance

Multiplex quantification of four DNA targets in one reaction with Bio-Rad droplet digital PCR system for GMO detection

The advantages of the digital PCR technology are already well documented until now. One way to achieve better cost efficiency of the technique is to use it in a multiplexing strategy. Droplet digital PCR platforms, which include two fluorescence filters, support at least duplex reactions and with some developments and optimization higher multiplexing is possible. The present study not only shows a development of multiplex assays in droplet digital PCR, but also presents a first thorough evaluation of several parameters in such multiplex digital PCR. Two 4-plex assays were developed for quantification of 8 different DNA targets (7 genetically modified maize events and maize endogene). Per assay, two of the targets were labelled with one fluorophore and two with another. As current analysis software does not support analysis of more than duplex, a new R- and Shiny-based web application analysis tool (http://bit.ly/ddPCRmulti) was developed that automates the analysis of 4-plex results. In conclusion, the two developed multiplex assays are suitable for quantification of GMO maize events and the same approach can be used in any other field with a need for accurate and reliable quantification of multiple DNA targets

Hysteroscopic sterilization with occlusion of sheep uterine tube using n-butyl-2-cyanoacrylate adhesive

Purpose: To evaluate the fertility and analyze the macroscopic, microscopic and morphometric aspects of sheep uterine tube sterilization with a hysteroscopically insert of n-butyl-2-cyanoacrylate adhesive. Methods: 12 adult sheep, with one previous pregnancy, were distributed as follows: group L (n=3) subjected to laparotomy and Pomeroy uterine tube ligation, group S (n=3) subjected to hysteroscopic application of saline solution in tube isthmus and group AD(n=6), that was subjected to hysteroscopic application of 0.5 ml of n-2-butil-cyanoacrylate in tube isthmus. They were mated with fertile males for ninety days. The non pregnant sheep, at the 90th day, were subjected to laparotomy with uterus and tubes uterine resection. The fragments of uterine tubes were fixated in 10% formalin and processes for histology evaluated, and slices dyes for H.E. Data were evaluated by Wilcoxon and Mann-Whitney and Fisher’s exact test. Results: All sheep from groups L and AD did not get pregnant (0%) in contrast with sheep from group S (100%); the adhesive remained integral in the uterine tube lumen. The percentual of adherences (66.6%) and fibrosis responses (100%) was significantly higher in the group L than group AD (0%) (p≤0.01). The diameter of the caudal tube in group AD (2652.15 ± 45.76 mm) was significantly wider than that of the group L (1868.27 ± 56.11* mm) (p ≤ 0.05). Conclusion: The hysteroscopic insertion of cyanoacrylate in the uterine tube lumen of sheep was effective to obstruct the uterine tube and to promote the sterilization.Objetivo: Avaliar a fertilidade e aspectos macroscópicos, microscópicos e morfométricos da esterilização histeroscópica de tubas uterinas de ovelhas com o adesivo de n-butil-2-cianoacrilato. Métodos: 12 ovelhas adultas, com uma prenhez anterior, foram distribuídas como segue: o grupo L (n=3) submetidas à laparotomia e laqueadura tipo Pomeroy, grupo S (n=3) submetidas à aplicação histeroscópica de solução salina no istmo tubário e grupo AD (n=6), com aplicação histeroscópica de 0,5 ml de cianoacrilato. As ovelhas foram acasaladas com machos de comprovada fertilidade por noventa dias. As ovelhas não prenhes aos 90 dias, foram submetidas à laparotomia com ressecção do útero e tubas uterinas, que foram fixadas em formalina 10%s e os cortes histológicos corados em hematoxilina/eosina. Os resultados foram avaliados pelo teste de Wilcoxon e teste exato de Fisher. Resultados: Todas as ovelhas dos grupos L e AD não ficaram prenhes (0%) ao contrário das ovelhas do grupo S (100%); o adesivo permaneceu íntegro no lúmen tubário. O percentual de aderências (66.6%) e de fibrose (100%) foi significativamente maior no grupo L do que no grupo AD (0%) (p≤0,01). O diâmetro da porção caudal no grupo AD (2652,15 ± 45,76 mm) foi significativamente maior do que grupo L (1868,27 ± 56.11 mm) (p≤0,05). Conclusão: A inserção histeroscópica do cianoacrilato no lúmen tubário de ovelhas foi eficaz para obstruir a tuba uterina e promover a esterilização