BackgroundThe gene desert on human chromosomal band 8q24 harbors multiple genetic variants associated with common cancers, including breast cancer. The locus, including the gene desert and its flanking genes, MYC, PVT1 and FAM84B, is also frequently amplified in human breast cancer. We generated a megadeletion (MD) mouse model lacking 430-Kb of sequence orthologous to the breast cancer-associated region in the gene desert. The goals were to examine the effect of the deletion on mammary cancer development and on transcript level regulation of the candidate genes within the locus.MethodsThe MD allele was engineered using the MICER system in embryonic stem cells and bred onto 3 well-characterized transgenic models for breast cancer, namely MMTV-PyVT, MMTV-neu and C3(1)-TAg. Mammary tumor growth, latency, multiplicity and metastasis were compared between homozygous MD and wild type mice carrying the transgenes. A reciprocal mammary gland transplantation assay was conducted to distinguish mammary cell-autonomous from non-mammary cell-autonomous anti-cancer effects. Gene expression analysis was done using quantitative real-time PCR. Chromatin interactions were evaluated by 3C. Gene-specific patient outcome data were analysed using the METABRIC and TCGA data sets through the cBioPortal website.ResultsMice homozygous for the MD allele are viable, fertile, lactate sufficiently to nourish their pups, but maintain a 10% lower body weight mainly due to decreased adiposity. The deletion interferes with mammary tumorigenesis in mouse models for luminal and basal breast cancer. In the MMTV-PyVT model the mammary cancer-reducing effects of the allele are mammary cell-autonomous. We found organ-specific effects on transcript level regulation, with Myc and Fam84b being downregulated in mammary gland, prostate and mammary tumor samples. Through analysis using the METABRIC and TCGA datasets, we provide evidence that MYC and FAM84B are frequently co-amplified in breast cancer, but in contrast with MYC, FAM84B is frequently overexpressed in the luminal subtype, whereas MYC activity affect basal breast cancer outcomes.ConclusionDeletion of a breast cancer-associated non-protein coding region affects mammary cancer development in 3 transgenic mouse models. We propose Myc as a candidate susceptibility gene, regulated by the gene desert locus, and a potential role for Fam84b in modifying breast cancer development.Electronic supplementary materialThe online version of this article (10.1186/s12885-018-5109-8) contains supplementary material, which is available to authorized users.

Anti-PD-1/PD-L1 immunotherapy could offer an alternative to traditional chemo-and/or radiotherapy to treat pediatric cancer patients. To unveil the potential benefit of this new therapeutic approach, the prevalence of PD-L1 and other relevant immune markers using quantitative digital image analysis (DIA) could help to clarify this point. A bridging study was first conducted using commercially available normal formalin-fixed paraffinembedded (FFPE) tonsils to compare immunostaining patterns and intensities from PD-L1, tumor infiltrating lymphocyte (TIL) markers CD3, CD8, FoxP3, CD45RO, and macrophage marker CD68 in adult (n = 5) and pediatric (n = 10) samples. Then, commercially available pediatric FFPE tumor samples from five prevalent pediatric solid tumor indications: ganglioneuroblastoma (n = 7); neuroblastoma (n = 23); nephroblastoma (n = 30); osteosarcoma (n = 24); and rhabdomyosarcoma (n = 25) were immunostained and their images (n = 654) digitally analyzed using predefined algorithms. The qualitative analysis of staining patterns and intensities in all 15 tonsils for all 6 biomarkers was similar regardless of age category. Quantitative DIA showed that PD-L1 values varied across cancer-types, nephroblastoma having the lowest counts. PD-L1 counts in ganglioneuroblastoma, our pediatric indication with the highest average value, was approximately 12-times lower than in a similar nonsmall cell lung cancer study, an indication approved for anti-PD-1/PD-L1 immunotherapies. Variable values were measured for the TIL markers CD3, CD8, and CD45RO. FoxP3 was scant across all indications. The macrophage marker CD68 showed highest values in ganglioneuroblastoma, with lowest levels in nephroblastoma. In conclusion, the low PD-L1 levels uncorrelated with TIL values from the present biomarker morphological study suggest that a PD-L1 immunohistochemistry patient selection strategy used for anti-PD-1/PD-L1 monotherapy in adult tumors may not succeed in these pediatric indications. Figure 5. Clustered heatmaps of the individual cases (y-axis) based on standardized expression levels of the individual markers across investigated indications. Red or blue indicates higher or lower than average levels, respectively. 132MA Silva et al

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With the rising incidence of breast cancer cases in the population, it is imperative to understand mechanisms of susceptibility. Genome-wide association studies (GWAS) have identified many variants associated with breast cancer susceptibility. These loci have low penetrance and are common in the population. Some of the alleles are associated with specific subtypes of breast cancer and/or associate specifically within ancestral populations. Variants in the TOX3/LOC643714 locus on human chromosomal band 16q12.1 are strongly associated with risk to estrogen receptor-positive (ER+) breast cancer in cohorts of women of European ancestry. Remarkably, replication of the association of these same variants with breast cancer risk in women of African American ancestry failed, but other variants in a more distal area of the locus were found to be associated with risk. The correlated polymorphisms comprising these variants exist in noncoding regions, providing the possibility that these polymorphisms alter gene expression. The most likely causal gene, TOX3, encodes a transcription factor that has previously been found to regulate estrogen receptor alpha (ERα)-responsive genes in breast cancer cells. We hypothesize that noncoding breast cancer-associated polymorphisms on 16q12.1 regulate TOX3, subsequently modifying risk of developing ER+ breast cancer. To genetically dissect this locus, we implemented the CRISPR-Cas9 genetic engineering system in the rat model organism. The rat is the preferred rodent model for ER+ breast cancer. We engineered an allelic series of mutations generating rats with deletions in the portion of the Tox3 locus orthologous to the human risk-associated regions. We obtained viable mutants across all genotypes, despite partial embryonic lethality in homozygous Tox3 knockouts. The mutants in the allelic series display variable levels of Tox3 downregulation in the mammary gland, suggesting the presence of multiple Tox3-regulatory elements. Mutants showing altered Tox3 expression also show significant effects on mammary gland development, implicating a role for Tox3 in mammary stem/progenitor cell biology potentially through ERα gene regulation. In accordance with the association of the human risk-increasing allele with lower TOX3 transcript level, preliminary data from mammary carcinogenesis experiments in our rat model indicate that lower Tox3 levels result in increased mammary carcinoma multiplicity. This result suggests that Tox3 is a breast cancer susceptibility gene. Interestingly, Tox3 knockout rats present an obesity phenotype, male and female sterility, and a behavioral phenotype (increased anxiety), indicating pleiotropic effects of TOX3 loss-of-function mutations. Ongoing studies are focused on Tox3 in rat mammary gland biology and carcinogenesis to elucidate the mechanism of Tox3 regulation in susceptibility to ER+ breast cancer. Understanding mechanisms of susceptibility genes will ultimately lead to innovative strategies aimed at preventing breast cancer. Citation Format: Lauren B. Shunkwiler, Royal Pipaliya, Cody C. Ashy, Benjamine Van Peel, Yang Zhao, Jan Guz, Alexander Awgulewitsch, Michael J. Kern, Bart M.G. Smits. An allelic series of rat mutations reveal a role of TOX3 in mammary gland development, obesity, and breast cancer susceptibility [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B29.

Non-protein coding regions have frequently been associated with cancer susceptibility, with the human 8q24 gene desert linked to decreased incidence of breast cancer. To examine the impact of 8q24 upon mammary tissue development and breast cancer dynamics, we created a novel megadeletion (MD) mouse model lacking 430kb of the region orthologous to the human 8q24 gene desert. Homozygous MD knockout (MD-/-) mice have lower MYC expression in multiple organs, including whole mammary gland and mammary tumors, 10% reduced body weight, and strongly reduced susceptibility to luminal and to a lesser degree basal breast cancer compared to wild type animals. The latter finding mirrors human genetic data that associates the 8q24 gene desert variant more strongly with Estrogen Receptor-positive (ER+) than ER-negative (ER-) disease. In this study, we found that the MD-/- alters mammary gland development, resulting in a decrease in ductal branch points, fewer terminal end buds, and lower luminal/basal ratio. Using a reciprocal mammary gland transplantation assay, we found a strong donor effect and weaker host effect of the mutation on mammary gland development, indicating that the non-protein coding locus affects mammary cell-autonomous as well as non-mammary cell-autonomous processes. Interestingly, MD-/- resulted in a marked decrease in immune cell infiltrate in mammary tissues during development and throughout spontaneous tumor induction driven via the PyMT oncogene. Cellular profiling demonstrated a significant loss of mammary tissue-resident macrophage populations, but not monocyte-derived macrophages, with concomitant polarization away from homeostatic, developmental phenotypes which may be subverted to support breast cancer tumorigenesis. Using an integrative computational approach combining flow cytometry-based cell phenotyping with RNAseq-derived differential gene expression, we identified gene set and motif enrichment networks from mammary epithelium samples from wild type and knockout mice regulating mammary gland development, cancer processes, and immune function including inflammatory processes in regions affected by the MD locus. Taken together, these findings suggest that 8q24-mediated disruption of homeostatic mammary macrophage populations, either via MYC or yet undetermined mechanisms, may deprive neoplastic cells of crucial support from the stroma, inhibiting cancer initiation and progression. Our findings not only elucidate how the murine ortholog of the 8q24 gene desert, and likely MYC expression regulation, alters mammary gland development, but also provides novel insight into potential links between mammary development, immune infiltration, and breast cancer susceptibility. Citation Format: Bart M. Smits, Anna I. Rissman, Lauren Shunkwiler, Collin Homer-Bouthiette, Yang Zhao, Benjamine van Peel, Jennifer Schulte, Robert Wilson, Adam C. Soloff. Absence of mammary tissue-resident macrophages is associated with reduced breast cancer susceptibility mediated by the cancer-associated 8q24 gene desert [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5007.