A major challenge to personalized oncology is that driver mutations vary among cancer cells inhabiting the same tumor. Whether this reflects principally disparate patterns of Darwinian evolution in different tumor regions has remained unexplored. We mapped the prevalence of genetically distinct clones over 250 regions in 54 childhood cancers. This showed that primary tumors can simultaneously follow up to four evolutionary trajectories over different anatomic areas. The most common pattern consists of subclones with very few mutations confined to a single tumor region. The second most common is a stable coexistence, over vast areas, of clones characterized by changes in chromosome numbers. This is contrasted by a third, less frequent, pattern where a clone with driver mutations or structural chromosome rearrangements emerges through a clonal sweep to dominate an anatomical region. The fourth and rarest pattern is the local emergence of a myriad of clones with TP53 inactivation. Death from disease was limited to tumors exhibiting the two last, most dynamic patterns.

Microarray data is subject to noise and systematic variation that negatively affects the resolution of copy number analysis. We describe Rawcopy, an R package for processing of Affymetrix CytoScan HD, CytoScan 750k and SNP 6.0 microarray raw intensities (CEL files). Noise characteristics of a large number of reference samples are used to estimate log ratio and B-allele frequency for total and allele-specific copy number analysis. Rawcopy achieves better signal-to-noise ratio and higher proportion of validated alterations than commonly used free and proprietary alternatives. In addition, Rawcopy visualizes each microarray sample for assessment of technical quality, patient identity and genome-wide absolute copy number states. Software and instructions are available at http://rawcopy.org.

Purpose: Undifferentiated uterine sarcomas (UUS) are rare, extremely deadly, sarcomas with no effective treatment. The goal of this study was to identify novel intrinsic molecular UUS subtypes using integrated clinical, histopathologic, and molecular evaluation of a large, fully annotated, patient cohort. Experimental Design: Fifty cases of UUS with full clinicopathologic annotation were analyzed for gene expression (n ¼ 50), copy-number variation (CNV, n ¼ 40), cell morphometry (n ¼ 39), and protein expression (n ¼ 22). Gene ontology and network enrichment analysis were used to relate over-and underexpressed genes to pathways and further to clinicopathologic and phenotypic findings. Results: Gene expression identified four distinct groups of tumors, which varied in their clinicopathologic parameters. Gene ontology analysis revealed differential activation of pathways related to genital tract development, extracellular matrix (ECM), muscle function, and proliferation. A multivariable, adjusted Cox proportional hazard model demonstrated that RNA group, mitotic index, and hormone receptor expression influence patient overall survival (OS). CNV arrays revealed characteristic chromosomal changes for each group. Morphometry demonstrated that the ECM group, the most aggressive, exhibited a decreased cell density and increased nuclear area. A cell density cutoff of 4,300 tumor cells per mm 2 could separate ECM tumors from the remaining cases with a sensitivity of 83% and a specificity of 94%. IHC staining of MMP-14, Collagens 1 and 6, and Fibronectin proteins revealed differential expression of these ECM-related proteins, identifying potential new biomarkers for this aggressive sarcoma subgroup. Conclusions: Molecular evaluation of UUS provides novel insights into the biology, prognosis, phenotype, and possible treatment of these tumors.