SUMMARY Beyond sample curation and basic pathologic characterization, the digitized H&E-stained images of TCGA samples remain underutilized. To highlight this resource, we present mappings of tumor-infiltrating lymphocytes (TILs) based on H&E images from 13 TCGA tumor types. These TIL maps are derived through computational staining using a convolutional neural network trained to classify patches of images. Affinity propagation revealed local spatial structure in TIL patterns and correlation with overall survival. TIL map structural patterns were grouped using standard histopathological parameters. These patterns are enriched in particular T cell subpopulations derived from molecular measures. TIL densities and spatial structure were differentially enriched among tumor types, immune subtypes, and tumor molecular subtypes, implying that spatial infiltrate state could reflect particular tumor cell aberration states. Obtaining spatial lymphocytic patterns linked to the rich genomic characterization of TCGA samples demonstrates one use for the TCGA image archives with insights into the tumor-immune microenvironment.

Summary RNA interference is a powerful tool for studying gene function, however, the reproducible generation of RNAi transgenic mice remains a significant limitation. By combining optimized fluorescence-coupled miR30-based shRNAs with high efficiency ES cell targeting, we developed a fast, scalable pipeline for the production of shRNA transgenic mice. Using this system, we generated eight tet-regulated shRNA transgenic lines targeting Firefly and Renilla luciferases, Oct4 and tumor suppressors p53, p16INK4a, p19ARF and APC and demonstrate potent gene silencing and GFP-tracked knockdown in a broad range of tissues in vivo. Further, using an shRNA targeting APC, we illustrate how this approach can identify predicted phenotypes and also unknown functions for a well-studied gene. In addition, through regulated gene silencing we validate APC/Wnt and p19ARF as potential therapeutic targets in T cell acute lymphoblastic leukemia/lymphoma and lung adenocarcinoma, respectively. This system provides a cost-effective and scalable platform for the production of RNAi transgenic mice targeting any mammalian gene.

The etiology and pathogenesis of the vascular lesions characterizing primary pulmonary hypertension (PPH), an often fatal pulmonary vascular disease, are largely unknown. Plexiform lesions composed of proliferating endothelial cells occur in between 20 and 80% of the cases of this irreversible pulmonary vascular disease. Recently, technology to assess monoclonality has allowed the distinction between cellular proliferation present in neoplasms from that in reactive nonneoplastic tissue. To determine whether the endothelial cell proliferation in plexiform lesions in PPH is monoclonal or polyclonal, we assessed the methylation pattern of the human androgen receptor gene by PCR (HUMARA) in proliferated endothelial cells in plexiform lesions from female PPH patients (n = 4) compared with secondary pulmonary hypertension (PH) patients (n = 4). In PPH, 17 of 22 lesions (77%) were monoclonal. However, in secondary PH, all 19 lesions examined were polyclonal. Smooth muscle cell hyperplasia in pulmonary vessels (n = 11) in PPH and secondary PH was polyclonal in all but one of the examined vessels. The monoclonal expansion of endothelial cells provides the first marker that allows the distinction between primary and secondary PH. Our data of a frequent monoclonal endothelial cell proliferation in PPH suggests that a somatic genetic alteration similar to that present in neoplastic processes may be responsible for the pathogenesis of PPH.

Summary Phosphoenolpyruvate carboxykinase (PEPCK) is well known for its role in gluconeogenesis. However, PEPCK is also a key regulator of TCA cycle flux. The TCA cycle integrates glucose, amino acid and lipid metabolism depending on cellular needs. In addition, biosynthetic pathways crucial to tumor growth require the TCA cycle for the processing of glucose and glutamine derived carbons. We show here an unexpected role for PEPCK in promoting cancer cell proliferation in vitro and in vivo by increasing glucose and glutamine utilization toward anabolic metabolism. Unexpectedly, PEPCK also increased the synthesis of ribose from non-carbohydrate sources, such as glutamine, a phenomenon not previously described. Finally, we show that the effects of PEPCK on glucose metabolism and cell proliferation are in part mediated via activation of mTORC1. Taken together, these data demonstrate a role for PEPCK that links metabolic flux and anabolic pathways to cancer cell proliferation.

Purpose: This study was designed to investigate the expression of B7-H4 protein, a member of the B7 family that is involved in the regulation of antigen-specific immune responses, in normal breast and in primary and metastatic breast carcinomas.Experimental Design: Archival formalin-fixed tissue blocks from breast cancers and normal somatic tissues were evaluated for B7-H4 expression by immunohistochemistry with manual and automated image analysis. The proportion of B7-H4-positive cells and the intensity of B7-H4 staining were compared with histologic type, grade, stage, hormone receptor status, and HER-2/neu status.Results: B7-H4 was detected in 165 of 173 (95.4%) primary breast cancers and in 240 of 246 (97.6%) metastatic breast cancers. B7-H4 staining intensity was greater in invasive ductal carcinomas [24.61 relative units (RU)] and in invasive lobular carcinomas (15.23 RU) than in normal breast epithelium (4.30 RU, P = 0.0003). Increased staining intensity was associated with negative progesterone receptor status (P = 0.014) and history of neoadjuvant chemotherapy (P = 0.004), and the proportion of B7-H4-positive cells was associated with negative progesterone receptor (P = 0.001) and negative HER-2/neu (P = 0.024) status. However, there was no statistically significant relationship between the proportion of B7-H4-positive cells or staining intensity and grade, stage, or other clinicopathologic variables. Low levels of B7-H4 expression were also detected in epithelial cells of the female genital tract, lung, pancreas, and kidney, but B7-H4 was generally absent in most other normal somatic tissues.Conclusions: The nearly ubiquitous expression of B7-H4 in breast cancer, independent of tumor grade or stage, suggests a critical role for this protein in breast cancer biology.