The claudin-low breast cancer subtype is defined by gene expression characteristics and encompasses a remarkably diverse range of breast tumors. Here, we investigate genomic, transcriptomic, and clinical features of claudin-low breast tumors. We show that claudin-low is not simply a subtype analogous to the intrinsic subtypes (basal-like, HER2-enriched, luminal A, luminal B and normal-like) as previously portrayed, but is a complex additional phenotype which may permeate breast tumors of various intrinsic subtypes. Claudin-low tumors are distinguished by low genomic instability, mutational burden and proliferation levels, and high levels of immune and stromal cell infiltration. In other aspects, claudin-low tumors reflect characteristics of their intrinsic subtype. Finally, we explore an alternative method for identifying claudin-low tumors and thereby uncover potential weaknesses in the established claudin-low classifier. In sum, these findings elucidate the heterogeneity in claudin-low breast tumors, and substantiate a re-definition of claudin-low as a cancer phenotype.

Breast cancer is a heterogenous disease with variability in tumor cells and in the surrounding tumor microenvironment (TME). Understanding the molecular diversity in breast cancer is critical for improving prediction of therapeutic response and prognostication. High-plex spatial profiling of tumors enables characterization of heterogeneity in the breast TME, which can holistically illuminate the biology of tumor growth, dissemination and, ultimately, response to therapy. The GeoMx Digital Spatial Profiler (DSP) enables researchers to spatially resolve and quantify proteins and RNA transcripts from tissue sections. The platform is compatible with both formalin-fixed paraffin-embedded and frozen tissues. RNA profiling was developed at the whole transcriptome level for human and mouse samples and protein profiling of 100-plex for human samples. Tissue can be optically segmented for analysis of regions of interest or cell populations to study biology-directed tissue characterization. The GeoMx Breast Cancer Consortium (GBCC) is composed of breast cancer researchers who are developing innovative approaches for spatial profiling to accelerate biomarker discovery. Here, the GBCC presents best practices for GeoMx profiling to promote the collection of high-quality data, optimization of data analysis and integration of datasets to advance collaboration and meta-analyses. Although the capabilities of the platform are presented in the context of breast cancer research, they can be generalized to a variety of other tumor types that are characterized by high heterogeneity.

Systematically investigating the scores of genes mutated in cancer and discerning disease drivers from inconsequential bystanders is a prerequisite for precision medicine but remains challenging. Here, we developed a somatic CRISPR/Cas9 mutagenesis screen to study 215 recurrent “long-tail” breast cancer genes, which revealed epigenetic regulation as a major tumor-suppressive mechanism. We report that components of the BAP1 and COMPASS-like complexes, including KMT2C/D, KDM6A, BAP1, and ASXL1/2 (“EpiDrivers”), cooperate with PIK3CAH1047R to transform mouse and human breast epithelial cells. Mechanistically, we find that activation of PIK3CAH1047R and concomitant EpiDriver loss triggered an alveolar-like lineage conversion of basal mammary epithelial cells and accelerated formation of luminal-like tumors, suggesting a basal origin for luminal tumors. EpiDriver mutations are found in ∼39% of human breast cancers, and ∼50% of ductal carcinoma in situ express casein, suggesting that lineage infidelity and alveogenic mimicry may significantly contribute to early steps of breast cancer etiology. Significance: Infrequently mutated genes comprise most of the mutational burden in breast tumors but are poorly understood. In vivo CRISPR screening identified functional tumor suppressors that converged on epigenetic regulation. Loss of epigenetic regulators accelerated tumorigenesis and revealed lineage infidelity and aberrant expression of alveogenesis genes as potential early events in tumorigenesis. This article is highlighted in the In This Issue feature, p. 2711

Ductal carcinoma in situ (DCIS) is a non-invasive type of breast cancer with highly variable potential of becoming invasive and affecting mortality. Currently, many patients with DCIS are overtreated due to the lack of specific biomarkers that distinguish low risk lesions from those with a higher risk of progression. In this study, we analyzed 57 pure DCIS and 313 invasive breast cancers (IBC) from different patients. Three levels of genomic data were obtained; gene expression, DNA methylation, and DNA copy number. We performed subtype stratified analyses and identified key differences between DCIS and IBC that suggest subtype specific progression. Prominent differences were found in tumors of the basal-like subtype: Basal-like DCIS were less proliferative and showed a higher degree of differentiation than basal-like IBC. Also, core basal tumors (characterized by high correlation to the basal-like centroid) were not identified amongst DCIS as opposed to IBC. At the copy number level, basal-like DCIS exhibited fewer copy number aberrations compared with basal-like IBC. An intriguing finding through analysis of the methylome was hypermethylation of multiple protocadherin genes in basal-like IBC compared with basal-like DCIS and normal tissue, possibly caused by long range epigenetic silencing. This points to silencing of cell adhesion-related genes specifically in IBC of the basal-like subtype. Our work confirms that subtype stratification is essential when studying progression from DCIS to IBC, and we provide evidence that basal-like DCIS show less aggressive characteristics and question the assumption that basal-like DCIS is a direct precursor of basal-like invasive breast cancer.

Breast cancers in humans belong to one of several intrinsic molecular subtypes each with different tumor biology and different clinical impact. Mammary gland tumors in dogs are proposed as a relevant comparative model for human breast cancer; however, it is still unclear whether the intrinsic molecular subtypes have the same significance in dogs and humans. Using publicly available data, we analyzed gene expression and whole-exome sequencing data from 158 canine mammary gland tumors. We performed molecular subtyping using the PAM50 method followed by subtype-specific comparisons of gene expression characteristics, mutation patterns and copy number profiles between canine tumors and human breast tumors from The Cancer Genome Atlas (TCGA) breast cancer cohort (n = 1097). We found that luminal A canine tumors greatly resemble luminal A human tumors both in gene expression characteristics, mutations and copy number profiles. Also, the basal-like canine and human tumors were relatively similar, with low expression of luminal epithelial markers and high expression of genes involved in cell proliferation. There were, however, distinct differences in immune-related gene expression patterns in basal-like tumors between the two species. Characteristic HER2-enriched and luminal B subtypes were not present in the canine cohort, and we found no tumors with high-level ERBB2 amplifications. Benign and malignant canine tumors displayed similar PAM50 subtype characteristics. Our findings indicate that deeper understanding of the different molecular subtypes in canine mammary gland tumors will further improve the value of canines as comparative models for human breast cancer.

Claudin-low breast cancer is a molecular subtype associated with poor prognosis and without targeted treatment options. The claudin-low subtype is defined by certain biological characteristics, some of which may be clinically actionable, such as high immunogenicity. In mice, the medroxyprogesterone acetate (MPA) and 7,12-dimethylbenzanthracene (DMBA)-induced mammary tumor model yields a heterogeneous set of tumors, a subset of which display claudin-low features. Neither the genomic characteristics of MPA/DMBA-induced claudin-low tumors nor those of human claudin-low breast tumors have been thoroughly explored. The transcriptomic characteristics and subtypes of MPA/DMBA-induced mouse mammary tumors were determined using gene expression microarrays. Somatic mutations and copy number aberrations in MPA/DMBA-induced tumors were identified from whole exome sequencing data. A publicly available dataset was queried to explore the genomic characteristics of human claudin-low breast cancer and to validate findings in the murine tumors. Half of MPA/DMBA-induced tumors showed a claudin-low-like subtype. All tumors carried mutations in known driver genes. While the specific genes carrying mutations varied between tumors, there was a consistent mutational signature with an overweight of T>A transversions in TG dinucleotides. Most tumors carried copy number aberrations with a potential oncogenic driver effect. Overall, several genomic events were observed recurrently; however, none accurately delineated claudin-low-like tumors. Human claudin-low breast cancers carried a distinct set of genomic characteristics, in particular a relatively low burden of mutations and copy number aberrations. The gene expression characteristics of claudin-low-like MPA/DMBA-induced tumors accurately reflected those of human claudin-low tumors, including epithelial-mesenchymal transition phenotype, high level of immune activation, and low degree of differentiation. There was an elevated expression of the immunosuppressive genes PTGS2 (encoding COX-2) and CD274 (encoding PD-L1) in human and murine claudin-low tumors. Our findings show that the claudin-low breast cancer subtype is not demarcated by specific genomic aberrations, but carries potentially targetable characteristics warranting further research.