Summary How organ-specific metastatic traits arise in primary tumors remains unknown. Here we show a role of the breast tumor stroma in selecting cancer cells that are primed for metastasis in bone. Cancer-associated fibroblasts (CAFs) in triple-negative (TN) breast tumors skew heterogeneous cancer cell populations towards a predominance of clones that thrive on the CAF-derived factors CXCL12 and IGF1. Limiting concentrations of these factors select for cancer cells with high Src activity, a known clinical predictor of bone relapse and an enhancer of PI3K-Akt pathway activation by CXCL12 and IGF1. Carcinoma clones selected in this manner are primed for metastasis in the CXCL12-rich microenvironment of the bone marrow. The evidence suggests that stromal signals resembling those of a distant organ select for cancer cells that are primed for metastasis in that organ, thus illuminating the evolution of metastatic traits in a primary tumor and its distant metastases.

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Purpose Metaplastic breast carcinoma (MBC) is a rare and aggressive histologic type of breast cancer predominantly of triple-negative phenotype, and characterized by the presence of malignant cells showing squamous and/or mesenchymal differentiation. We sought to define the repertoire of somatic genetic alterations and the mutational signatures of MBCs. Experimental Design Whole-exome sequencing was performed in 35 MBCs, with 16, ten and nine classified as harboring chondroid, spindle and squamous metaplasia as the predominant metaplastic component. The genomic landscape of MBCs was compared to that of triple-negative invasive ductal carcinomas of no special type (IDC-NSTs) from The Cancer Genome Atlas. Wnt and PI3K/AKT/mTOR pathway activity was assessed using a quantitative PCR assay. Results MBCs harbored complex genomes with frequent TP53 (69%) mutations. In contrast to triple-negative IDC-NSTs, MBCs more frequently harbored mutations in PIK3CA (29%), PIK3R1 (11%), ARID1A (11%), FAT1 (11%) and PTEN (11%). PIK3CA mutations were not found in MBCs with chondroid metaplasia. Compared to triple-negative IDC-NSTs, MBCs significantly more frequently harbored mutations in PI3K/AKT/mTOR pathway-related (57% vs 22%) and canonical Wnt pathway-related (51% vs 28%) genes. MBCs with somatic mutations in PI3K/AKT/mTOR or Wnt pathway-related genes displayed increased activity of the respective pathway. Conclusion MBCs are genetically complex and heterogeneous, and are driven by a repertoire of somatic mutations distinct from that of triple-negative IDC-NSTs. Our study highlights the genetic basis and the importance of PI3K/AKT/mTOR and Wnt pathway dysregulation in MBCs, and provides a rationale for the metaplastic phenotype and the reported responses to PI3K/AKT/mTOR inhibitors in these tumors.

Purpose To assess the safety and tolerability of pre-operative cryoablation-mediated tumor antigen presentation and/or ipilimumab-mediated immune modulation in women with operable breast cancer. Experimental design In this pilot study, 19 women with breast cancer for whom mastectomy was planned were treated with pre-operative tumor cryoablation (n=7), single-dose ipilimumab at 10mg/kg (n=6), or both (n=6). The primary outcome for this pilot study was safety/tolerability as defined as freedom from delays in pre-planned, curative-intent mastectomy. Exploratory studies of immune activation were performed on peripheral blood and tumor. Results Pre-operative cryoablation and/or ipilimumab were safe and tolerable, with no delays in pre-planned surgery. Grade III toxicity was seen in 1/19 (unrelated rash after ipilimumab). Combination therapy was associated with sustained peripheral elevations in: Th1-type cytokines, activated (ICOS+) and proliferating (Ki67+) CD4+ and CD8+ T cells, and post-treatment proliferative T-effector cells relative to T-regulatory cells within tumor. Conclusions Pre-operative cryoablation and single-dose ipilimumab are safe alone or in combination with no surgical delays incurred. Potentially favorable intra-tumoral and systemic immunologic effects were observed with the combination, suggesting the possibility for induced and synergistic anti-tumor immunity with this strategy.

Adenoid cystic carcinoma (AdCC) is a rare type of triple-negative breast cancer (TNBC) characterized by the presence of the MYB-NFIB fusion gene. The molecular underpinning of breast AdCCs other than the MYB-NFIB fusion gene remains largely unexplored. Here we sought to define the repertoire of somatic genetic alterations of breast AdCCs. We performed whole exome sequencing, followed by orthogonal validation, of 12 breast AdCCs to determine the landscape of somatic mutations and gene copy number alterations. Fluorescence in situ hybridization and reverse transcription PCR were used to define the presence of MYB gene rearrangements and MYB-NFIB chimeric transcripts. Unlike common forms of TNBC, we found that AdCCs have a low mutation rate (0.27 non-silent mutations/Mb), lack mutations in TP53 and PIK3CA, and display a heterogeneous constellation of known cancer genes affected by somatic mutations, including MYB, BRAF, FBXW7, SMARCA5, SF3B1 and FGFR2. MYB and TLN2 were affected by somatic mutations in two cases each. Akin to salivary gland AdCCs, breast AdCCs were found to harbor mutations targeting chromatin remodeling, cell adhesion, RNA biology, ubiquitination, and canonical signaling pathway genes. We observed that although breast AdCCs had rather simple genomes, they likely display intra-tumor genetic heterogeneity at diagnosis. Taken together, these findings demonstrate that the mutational burden and mutational repertoire of breast AdCCs are more similar to those of salivary gland AdCCs than to those of other types of TNBCs, emphasizing the importance of histologic subtyping of TNBCs. Furthermore, our data provide direct evidence that AdCCs harbor a distinctive mutational landscape and genomic structure, irrespective of disease site of origin.