Pitfalls in TCR gene clonality testing: teaching cases

Clonality testing in T-lymphoproliferations has technically become relatively easy to perform in routine laboratories using standardized multiplex polymerase chain reaction protocols for T-cell receptor (TCR) gene analysis as developed by the BIOMED-2 Concerted Action BMH4-CT98-3936. Expertise with clonality diagnostics and knowledge about the biology of TCR gene recombination are essential for correct interpretation of TCR clonality data. Several immunobiological and technical pitfalls that should be taken into account to avoid misinterpretation of data are addressed in this report. Furthermore, we discuss the need to integrate the molecular data with those from immunohistology, and preferably also flow cytometric immunophenotyping, for appropriate interpretation. Such an interactive, multidisciplinary diagnostic model guarantees integration of available data to reach the most reliable diagnosis

Detecting microRNA binding and siRNA off-target effects from expression data.

Sylamer is a method for detecting microRNA target and small interfering RNA off-target signals in 3' untranslated regions from a ranked gene list, sorted from upregulated to downregulated, after a microRNA perturbation or RNA interference experiment. The output is a landscape plot that tracks occurrence biases using hypergeometric P-values for all words across the gene ranking. We demonstrated the utility, speed and accuracy of this approach on several datasets

Human Papillomavirus (HPV) in breast tumors: prevalence in a group of Mexican patients

<p>Abstract</p> <p>Background</p> <p>Breast cancer is one of the main health problems in developed countries, occupying first place in mortality in women. It is well-known that there are risk factors associated with breast cancer development. Nonetheless, in 50–80% of cases known risk factors have not been identified, this has generated the attempt to identify new factors related with this neoplasia as viral infections. The aim of this work is investigate the prevalence of HPV DNA in patients with breast lesions at the Instituto Nacional de Cancerologia de Mexico.</p> <p>Methods</p> <p>Fifty-one cases of breast cancer were selected from the files of the institute and compared by age and tumor size with 43 cases of non malignant breast lesions (fibroadenoma, fibrocystic disease and phyllodes tumor). Paraffin embedded specimens were selected, HPV DNA was analyzed by polymerase chain reaction (PCR) and sequenced for different types of HPV in case of positivity for HPV-DNA. Descriptive analysis of clinical and pathological variables was performed and comparisons between positive and negative cases was done.</p> <p>Results</p> <p>All patients were mexican, mean age was 53.3, median age of menarche was 13 and median tumor size 9 cms. Cervicovaginal cytology was performed to all patients, 1 patient (1.9%) of cancer group had HPV and none in the other group, no cases were diagnosed with cervical dysplasia. In the group of carcinomas 36 (70.5%) were negative and 15 (29.4%) were positive to HPV-DNA, 10(66.6%) were positive for HPV 16, 3(20%) for HPV 18, two cases (13.4%) were positive for both. In the group of benign conditions all were negative to HPV-DNA.</p> <p>Conclusion</p> <p>Presence of HPV in breast cancer in our group of cases is high in comparison to other authors; larger numbers of cases need to be analyzed in order to establish the exact role of this virus in the pathogenesis of breast cancer.</p

Lack of correlation between predicted and actual off-target effects of short-interfering RNAs targeting the human papillomavirus type 16 E7 oncogene.

BACKGROUND: When designing therapeutic short-interfering RNAs (siRNAs), off-target effects (OTEs) are usually predicted by computational quantification of messenger RNAs (mRNAs) that contain matches to the siRNA seed sequence in their 3' UTRs. It is assumed that the higher the number of predicted transcriptional OTEs, the greater the size of the actual OTE signature and the more detrimental the phenotypic consequences in target-negative cells. METHODS: We tested this general assumption by investigating the OTEs of potential therapeutic siRNAs targeting the human papillomavirus (HPV) type-16 E7 oncogene. We studied HPV-negative squamous epithelial cells, from normal cervix (NCx) and skin (HaCaT), which would be vulnerable to 'bystander' OTEs following transfection in vivo. RESULTS: We observed no correlation between the number of computationally predicted OTEs and the actual number of seed-dependent OTEs (P=0.76). On average only 20.5% of actual transcriptional OTEs were seed-dependent (i.e., predicted). The unpredicted OTEs included stimulation of innate immune pathways, as well as indirect (downstream) effects of other OTEs, which affected important cancer-associated pathways. Although most significant OTEs observed were seen in both NCx and HaCaT cells, only 0-5.9% of differentially expressed genes overlapped between the two cell types. CONCLUSION: These data do not support the assumption that actual OTEs correlate well with predicted OTEs

Activating mutations of the GNAQ gene: a frequent event in primary melanocytic neoplasms of the central nervous system

Primary melanocytic neoplasms of the central nervous system (CNS) are uncommon neoplasms derived from melanocytes that normally can be found in the leptomeninges. They cover a spectrum of malignancy grades ranging from low-grade melanocytomas to lesions of intermediate malignancy and overtly malignant melanomas. Characteristic genetic alterations in this group of neoplasms have not yet been identified. Using direct sequencing, we investigated 19 primary melanocytic lesions of the CNS (12 melanocytomas, 3 intermediate-grade melanocytomas, and 4 melanomas) for hotspot oncogenic mutations commonly found in melanocytic tumors of the skin (BRAF, NRAS, and HRAS genes) and uvea (GNAQ gene). Somatic mutations in the GNAQ gene at codon 209, resulting in constitutive activation of GNAQ, were detected in 7/19 (37%) tumors, including 6/12 melanocytomas, 0/3 intermediate-grade melanocytomas, and 1/4 melanomas. These GNAQ-mutated tumors were predominantly located around the spinal cord (6/7). One melanoma carried a BRAF point mutation that is frequently found in cutaneous melanomas (c.1799 T>A, p.V600E), raising the question whether this is a metastatic rather than a primary tumor. No HRAS or NRAS mutations were detected. We conclude that somatic mutations in the GNAQ gene at codon 209 are a frequent event in primary melanocytic neoplasms of the CNS. This finding provides new insight in the pathogenesis of these lesions and suggests that GNAQ-dependent mitogen-activated kinase signaling is a promising therapeutic target in these tumors. The prognostic and predictive value of GNAQ mutations in primary melanocytic lesions of the CNS needs to be determined in future studies

Keeping Pace with Your Eating: Visual Feedback Affects Eating Rate in Humans

Deliberately eating at a slower pace promotes satiation and eating quickly has been associated with a higher body mass index. Therefore, understanding factors that affect eating rate should be given high priority. Eating rate is affected by the physical/textural properties of a food, by motivational state, and by portion size and palatability. This study explored the prospect that eating rate is also influenced by a hitherto unexplored cognitive process that uses ongoing perceptual estimates of the volume of food remaining in a container to adjust intake during a meal. A 2 (amount seen; 300ml or 500ml) x 2 (amount eaten; 300ml or 500ml) between-subjects design was employed (10 participants in each condition). In two ‘congruent’ conditions, the same amount was seen at the outset and then subsequently consumed (300ml or 500ml). To dissociate visual feedback of portion size and actual amount consumed, food was covertly added or removed from a bowl using a peristaltic pump. This created two additional ‘incongruent’ conditions, in which 300ml was seen but 500ml was eaten or vice versa. We repeated these conditions using a savoury soup and a sweet dessert. Eating rate (ml per second) was assessed during lunch. After lunch we assessed fullness over a 60-minute period. In the congruent conditions, eating rate was unaffected by the actual volume of food that was consumed (300ml or 500ml). By contrast, we observed a marked difference across the incongruent conditions. Specifically, participants who saw 300ml but actually consumed 500ml ate at a faster rate than participants who saw 500ml but actually consumed 300ml. Participants were unaware that their portion size had been manipulated. Nevertheless, when it disappeared faster or slower than anticipated they adjusted their rate of eating accordingly. This suggests that the control of eating rate involves visual feedback and is not a simple reflexive response to orosensory stimulatio

EVA: Laparoscopic instrument tracking based on endoscopic video analysis for psychomotor skills assessment

INTRODUCTION: The EVA (Endoscopic Video Analysis) tracking system a new tracking system for extracting motions of laparoscopic instruments based on non-obtrusive video tracking was developed. The feasibility of using EVA in laparoscopic settings has been tested in a box trainer setup. METHODS: EVA makes use of an algorithm that employs information of the laparoscopic instrument's shaft edges in the image, the instrument's insertion point, and the camera's optical centre to track the 3D position of the instrument tip. A validation study of EVA comprised a comparison of the measurements achieved with EVA and the TrEndo tracking system. To this end, 42 participants (16 novices, 22 residents, and 4 experts) were asked to perform a peg transfer task in a box trainer. Ten motion-based metrics were used to assess their performance. RESULTS: Construct validation of the EVA has been obtained for seven motion-based metrics. Concurrent validation revealed that there is a strong correlation between the results obtained by EVA and the TrEndo for metrics such as path length (p=0,97), average speed (p=0,94) or economy of volume (p=0,85), proving the viability of EVA. CONCLUSIONS: EVA has been successfully used in the training setup showing potential of endoscopic video analysis to assess laparoscopic psychomotor skills. The results encourage further implementation of video tracking in training setups and in image guided surgery

Learning curves of basic laparoscopic psychomotor skills in SINERGIA VR simulator

Purpose: Surgical simulators are currently essential within any laparoscopic training program because they provide a low-stakes, reproducible and reliable environment to acquire basic skills. The purpose of this study is to determine the training learning curve based on different metrics corresponding to five tasks included in SINERGIA laparoscopic virtual reality simulator. Methods: Thirty medical students without surgical experience participated in the study. Five tasks of SINERGIA were included: Coordination, Navigation, Navigation and touch, Accurate grasping and Coordinated pulling. Each participant was trained in SINERGIA. This training consisted of eight sessions (R1–R8) of the five mentioned tasks and was carried out in two consecutive days with four sessions per day. A statistical analysis was made, and the results of R1, R4 and R8 were pair-wise compared with Wilcoxon signed-rank test. Significance is considered at P value <0.005. Results: In total, 84.38% of the metrics provided by SINERGIA and included in this study show significant differences when comparing R1 and R8. Metrics are mostly improved in the first session of training (75.00% when R1 and R4 are compared vs. 37.50% when R4 and R8 are compared). In tasks Coordination and Navigation and touch, all metrics are improved. On the other hand, Navigation just improves 60% of the analyzed metrics. Most learning curves show an improvement with better results in the fulfillment of the different tasks. Conclusions: Learning curves of metrics that assess the basic psychomotor laparoscopic skills acquired in SINERGIA virtual reality simulator show a faster learning rate during the first part of the training. Nevertheless, eight repetitions of the tasks are not enough to acquire all psychomotor skills that can be trained in SINERGIA. Therefore, and based on these results together with previous works, SINERGIA could be used as training tool with a properly designed training program