SUMMARY: Clinical sequencing aims to identify somatic mutations in cancer cells for accurate diagnosis and treatment. However, most widely used clinical assays lack patient-matched control DNA and additional analysis is needed to distinguish somatic and unfiltered germline variants. Such computational analyses require accurate assessment of tumor cell content in individual specimens. Histological estimates often do not corroborate with results from computational methods that are primarily designed for normal-tumor matched data and can be confounded by genomic heterogeneity and presence of sub-clonal mutations. Allele-frequency-based imputation of tumor (All-FIT) is an iterative weighted least square method to estimate specimen tumor purity based on the allele frequencies of variants detected in high-depth, targeted, clinical sequencing data. Using simulated and clinical data, we demonstrate All-FIT's accuracy and improved performance against leading computational approaches, highlighting the importance of interpreting purity estimates based on expected biology of tumors. AVAILABILITY AND IMPLEMENTATION: Freely available at http://software.khiabanian-lab.org. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The presence of lymphocytic infiltration (LI) has been correlated with nodal metastasis and tumor recurrence in HER2+breast cancer (BC), making it important to study LI. The ability to detect and quantify extent of LI could serve as an image based prognostic tool for HER2+ BC patients. Lymphocyte segmentation in H & E-stained BC histopathology images is, however, complicated due to the similarity in appearance between lymphocyte nuclei and cancer nuclei. Additional challenges include biological variability, histological artifacts, and high prevalence of overlapping objects. Although active contours are widely employed in segmentation, they are limited in their ability to segment overlapping objects. In this paper, we propose a segmentation scheme (EMaGACOR) that integrates Expectation Maximization (EM) based segmentation with a geodesic active contour (GAC). Additionally, a novel heuristic edge-path algorithm exploits the size of lymphocytes to split contours that enclose overlapping objects. For a total of 62 HER2+ breast biopsy images, EMaGACOR was found to have a detection sensitivity of over 90% and a positive predictive value of over 78%. By comparison, EMaGAC (model without overlap resolution) and GAC (Randomly initialized geodesic active contour) model yielded corresponding sensitivities of 57.4% and 26.7%, respectively. Furthermore, EMaGACOR was able to resolve over 92% of overlaps. Our scheme was found to be robust, reproducible, accurate, and could potentially be applied to other biomedical image segmentation applications.

BACKGROUND: Clustering analysis of microarray data is often criticized for giving ambiguous results because of sensitivity to data perturbation or clustering techniques used. In this paper, we describe a new method based on principal component analysis and ensemble consensus clustering that avoids these problems. RESULTS: We illustrate the method on a public microarray dataset from 36 breast cancer patients of whom 31 were diagnosed with at least two of three pathological stages of disease (atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC). Our method identifies an optimum set of genes and divides the samples into stable clusters which correlate with clinical classification into Luminal, Basal-like and Her2+ subtypes. Our analysis reveals a hierarchical portrait of breast cancer progression and identifies genes and pathways for each stage, grade and subtype. An intriguing observation is that the disease phenotype is distinguishable in ADH and progresses along distinct pathways for each subtype. The genetic signature for disease heterogeneity across subtypes is greater than the heterogeneity of progression from DCIS to IDC within a subtype, suggesting that the disease subtypes have distinct progression pathways. Our method identifies six disease subtype and one normal clusters. The first split separates the normal samples from the cancer samples. Next, the cancer cluster splits into low grade (pathological grades 1 and 2) and high grade (pathological grades 2 and 3) while the normal cluster is unchanged. Further, the low grade cluster splits into two subclusters and the high grade cluster into four. The final six disease clusters are mapped into one Luminal A, three Luminal B, one Basal-like and one Her2+. CONCLUSION: We confirm that the cancer phenotype can be identified in early stage because the genes altered in this stage progressively alter further as the disease progresses through DCIS into IDC. We identify six subtypes of disease which have distinct genetic signatures and remain separated in the clustering hierarchy. Our findings suggest that the heterogeneity of disease across subtypes is higher than the heterogeneity of the disease progression within a subtype, indicating that the subtypes are in fact distinct diseases.