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SUMMARYDuctal carcinoma in situ (DCIS) is the most common precursor of invasive breast cancer (IBC), with variable propensity for progression. We have performed the first multiscale, integrated profiling of DCIS with clinical outcomes by analyzing 677 DCIS samples from 481 patients with 7.1 years median follow-up from the Translational Breast Cancer Research Consortium (TBCRC) 038 study and the Resource of Archival Breast Tissue (RAHBT) cohorts. We identified 812 genes associated with ipsilateral recurrence within 5 years from treatment and developed a classifier that was predictive of DCIS or IBC recurrence in both cohorts. Pathways associated with recurrence include proliferation, immune response, and metabolism. Distinct stromal expression patterns and immune cell compositions were identified. Our multiscale approach employed in situ methods to generate a spatially resolved atlas of breast precancers, where complementary modalities can be directly compared and correlated with conventional pathology findings, disease states, and clinical outcome.HIGHLIGHTS⍰ Development of a new classifier for DCIS recurrence or progression⍰ Outcome associated pathways identified across multiple data types and compartments⍰ Four stroma-specific signatures identified⍰ CNAs characterize DCIS subgroups associated with high risk invasive cancers

Ductal carcinomain situ(DCIS) is a pre-invasive lesion that is thought to be a precursor to invasive breast cancer (IBC). To understand how the tumor microenvironment (TME) changes with transition to IBC, we used Multiplexed Ion Beam Imaging by time of flight (MIBI-TOF) and a 37-plex antibody staining panel to analyze 140 clinically annotated surgical resections covering the full spectrum of breast cancer progression. We compared normal, DCIS, and IBC tissues using machine learning tools for multiplexed cell segmentation, pixel-based clustering, and object morphometrics. Transition from DCIS to IBC was found to occur along a trajectory marked by coordinated shifts in location and function of myoepithelium, fibroblasts, and infiltrating immune cells in the surrounding stroma. Taken together, this comprehensive study within the HTAN Breast PreCancer Atlas offers insight into the etiologies of DCIS, its transition to IBC, and emphasizes the importance of the TME stroma in promoting these processes.

Tumors comprise a complex microenvironment of interacting malignant and stromal cell types. Much of our understanding of the tumor microenvironment comes from in vitro studies isolating the interactions between malignant cells and a single stromal cell type, often along a single pathway. To develop a deeper understanding of the interactions between cells within human lung tumors we performed RNA-seq profiling of flow-sorted malignant cells, endothelial cells, immune cells, fibroblasts, and bulk cells from freshly resected human primary non-small-cell lung tumors. We mapped the cell-specific differential expression of prognostically-associated secreted factors and cell surface genes, and computationally reconstructed cross-talk between these cell types to generate a novel resource we call the Lung Tumor Microenvironment Interactome (LTMI). Using this resource, we identified and validated a prognostically unfavorable influence of Gremlin-1 production by fibroblasts on proliferation of malignant lung adenocarcinoma cells. We also found a prognostically favorable association between infiltration of mast cells and less aggressive tumor cell behavior. These results illustrate the utility of the LTMI as a resource for generating hypotheses concerning tumor-microenvironment interactions that may have prognostic and therapeutic relevance.SummaryRNA-seq profiling of sorted populations from primary lung cancer samples identifies prognostically relevant cross-talk between cell types in the tumor microenvironment.

We present the discovery of genes recurrently involved in structural variation in nasopharyngeal carcinoma (NPC) and the identification of a novel type of somatic structural variant. We identified the variants with high complexity mate-pair libraries and a novel computational algorithm specifically designed for tumor-normal comparisons, SMASH. SMASH combines signals from split reads and mate-pair discordance to detect somatic structural variants. We demonstrate a >90% validation rate and a breakpoint reconstruction accuracy of 3 bp by Sanger sequencing. Our approach identified three in-frame gene fusions (YAP1-MAML2, PTPLB-RSRC1, and SP3-PTK2) that had strong levels of expression in corresponding NPC tissues. We found two cases of a novel type of structural variant, which we call ‘‘coupled inversion,’’ one of which produced the YAP1-MAML2 fusion. To investigate whether the identified fusion genes are recurrent, we performed fluorescent in situ hybridization (FISH) to screen 196 independent NPC cases. We observed recurrent rearrangements of MAML2 (three cases), PTK2 (six cases), and SP3 (two cases), corresponding to a combined rate of structural variation recurrence of 6% among tested NPC tissues. [Supplemental material is available for this article.] Nasopharyngeal carcinoma (NPC) is a malignant neoplasm of the head and neck originating in the epithelial lining of the nasopharynx. It has a high incidence among the native people of the American Arctic and Greenland and in southern Asia (Yu and Yuan 2002). NPC is strongly linked to consumption of Cantonese salted fish (Ning et al. 1990) and infection with Epstein-Barr virus (EBV) (Raab-Traub 2002), which is almost invariably present within the