Using a priori knowledge to align sequencing reads to their exact genomic position

The use of a priori knowledge in the alignment of targeted sequencing data is investigated using computational experiments. Adapting a Needleman–Wunsch algorithm to incorporate the genomic position information from the targeted capture, we demonstrate that alignment can be done to just the target region of interest. When in addition use is made of direct string comparison, an improvement of up to a factor of 8 in alignment speed compared to the fastest conventional aligner (Bowtie) is obtained. This results in a total alignment time in targeted sequencing of around 7 min for aligning approximately 56 million captured reads. For conventional aligners such as Bowtie, BWA or MAQ, alignment to just the target region is not feasible as experiments show that this leads to an additional 88% SNP calls, the vast majority of which are false positives (∼92%)

ER and PI3K pathway activity in primary ER positive breast cancer is associated with progression-free survival of metastatic patients under first-line tamoxifen

Estrogen receptor positive (ER+) breast cancer patients are eligible for hormonal treatment, but only around half respond. A test with higher specificity for prediction of endocrine therapy response is needed to avoid hormonal overtreatment and to enable selection of alternative treatments. A novel testing method was reported before that enables measurement of functional signal transduction pathway activity in individual cancer tissue samples, using mRNA levels of target genes of the respective pathway-specific transcription factor. Using this method, 130 primary breast cancer samples were analyzed from non-metastatic ER+ patients, treated with surgery without adjuvant hormonal therapy, who subsequently developed metastatic disease that was treated with first-line tamoxifen. Quantitative activity levels were measured of androgen and estrogen receptor (AR and ER), PI3K-FOXO, Hedgehog (HH), NFκB, TGFβ, and Wnt pathways. Based on samples with known pathway activity, thresholds were set to distinguish low from high activity. Subsequently, pathway activity levels were correlated with the tamoxifen treatment response and progression-free survival. High ER pathway activity was measured in 41% of the primary tumors and was associated with longer time to progression (PFS) of metastases during first-line tamoxifen treatment. In contrast, high PI3K, HH, and androgen receptor pathway activity was associated with shorter PFS, and high PI3K and TGFβ pathway activity with worse treatment response. Potential clinical utility of assessment of ER pathway activity lies in predicting response to hormonal therapy, while activity of PI3K, HH, TGFβ, and AR pathways may indicate failure to respond, but also opens new avenues for alternative or complementary targeted treatments

RNA based approaches to profile oncogenic pathways from low quantity samples to drive precision oncology strategies

Precision treatment of cancer requires knowledge on active tumor driving signal transduction pathways to select the optimal effective targeted treatment. Currently only a subset of patients derive clinical benefit from mutation based targeted treatment, due to intrinsic and acquired drug resistance mechanisms. Phenotypic assays to identify the tumor driving pathway based on protein analysis are difficult to multiplex on routine pathology samples. In contrast, the transcriptome contains information on signaling pathway activity and can complement genomic analyses. Here we present the validation and clinical application of a new knowledge-based mRNA-based diagnostic assay platform (OncoSignal) for measuring activity of relevant signaling pathways simultaneously and quantitatively with high resolution in tissue samples and circulating tumor cells, specifically with very small specimen quantities. The approach uses mRNA levels of a pathway\u27s direct target genes, selected based on literature for multiple proof points, and used as evidence that a pathway is functionally activated. Using these validated target genes, a Bayesian network model has been built and calibrated on mRNA measurements of samples with known pathway status, which is used next to calculate a pathway activity score on individual test samples. Translation to RT-qPCR assays enables broad clinical diagnostic applications, including small analytes. A large number of cancer samples have been analyzed across a variety of cancer histologies and benchmarked across normal controls. Assays have been used to characterize cell types in the cancer cell microenvironment, including immune cells in which activated and immunotolerant states can be distinguished. Results support the expectation that the assays provide information on cancer driving signaling pathways which is difficult to derive from next generation DNA sequencing analysis. Current clinical oncology applications have been complementary to genomic mutation analysis to improve precision medicine: (1) prediction of response and resistance to various therapies, especially targeted therapy and immunotherapy; (2) assessment and monitoring of therapy efficacy; (3) prediction of invasive cancer cell behavior and prognosis; (4) measurement of circulating tumor cells. Preclinical oncology applications lie in a better understanding of cancer behavior across cancer types, and in development of a pathophysiology-based cancer classification for development of novel therapies and precision medicine

Heterogeneity in signaling pathway activity within primary and between primary and metastatic breast cancer

Targeted therapy aims to block tumor-driving signaling pathways and is generally based on analysis of one primary tumor (PT) biopsy. Tumor heterogeneity within PT and between PT and metastatic breast lesions may, however, impact the effect of a chosen therapy. Whereas studies are available that investigate genetic heterogeneity, we present results on phenotypic heterogeneity by analyzing the variation in the functional activity of signal transduction pathways, using an earlier developed platform to measure such activity from mRNA measurements of pathways’ direct target genes. Statistical analysis comparing macro-scale variation in pathway activity on up to five spatially distributed PT tissue blocks (n = 35), to micro-scale variation in activity on four adjacent samples of a single PT tissue block (n = 17), showed that macro-scale variation was not larger than micro-scale variation, except possibly for the PI3K pathway. Simulations using a “checkerboard clone-size” model showed that multiple small clones could explain the higher micro-scale variation in activity found for the TGFβ and Hedgehog pathways, and that intermediate/large clones could explain the possibly higher macro-scale variation of the PI3K pathway. While within PT, pathway activities presented a highly positive correlation, correlations weakened between PT and lymph node metastases (n = 9), becoming even worse for PT and distant metastases (n = 9), including a negative correlation for the ER pathway. While analysis of multiple sub-samples of a single biopsy may be sufficient to predict PT response to targeted therapies, metastatic breast cancer treatment prediction requires analysis of metastatic biopsies. Our findings on phenotypic intra-tumor heterogeneity are compatible with emerging ideas on a Big Bang type of cancer evolution in which macro-scale heterogeneity appears not dominant

On randomness of random number generators

Many computer simulations use random number generators and since most computer languages have a built-in generator it is very easy just to use that one. However these random number generators can be very non-random

Optimising Attribute Selection in Conversational Search

It has been shown that user modelling has the potential to improve the performance of conversational search systems, particularly in what concerns the problem of attribute selection, i.e., determining which attribute to ask the user at each step of the dialogue. In this paper we present a novel framework for attribute selection which allows the fine-tuning of the relative importance of profile-based and entropy-based heuristics. Based on this framework, we describe a number of experiments which allow us to quantify the bounds to such improvements

Multidimensional Periodic Scheduling

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