Neoadjuvant and adjuvant chemotherapies provide survival benefits to breast cancer patients, in particular in estrogen receptor negative (ER − ) cancers, by reducing rates of recurrences. It is assumed that the benefits of (neo)adjuvant chemotherapy are due to the killing of disseminated, residual cancer cells, however, there is no formal evidence for it. Here, we provide experimental evidence that ER − breast cancer cells that survived high-dose Doxorubicin and Methotrexate based chemotherapies elicit a state of immunological dormancy. Hallmark of this dormant phenotype is the sustained activation of the IRF7/IFN-β/IFNAR axis subsisting beyond chemotherapy treatment. Upregulation of IRF7 in treated cancer cells promoted resistance to chemotherapy, reduced cell growth and induced switching of the response from a myeloid derived suppressor cell-dominated immune response to a CD4 + /CD8 + T cell-dependent anti-tumor response. IRF7 silencing in tumor cells or systemic blocking of IFNAR reversed the state of dormancy, while spontaneous escape from dormancy was associated with loss of IFN-β production. Presence of IFN-β in the circulation of ER − breast cancer patients treated with neoadjuvant Epirubicin chemotherapy correlated with a significantly longer distant metastasis-free survival. These findings establish chemotherapy-induced immunological dormancy in ER − breast cancer as a novel concept for (neo)adjuvant chemotherapy activity, and implicate sustained activation of the IRF7/IFN-β/IFNAR pathway in this effect. Further, IFN-β emerges as a potential predictive biomarker and therapeutic molecule to improve outcome of ER − breast cancer patients treated with (neo)adjuvant chemotherapy.

BACKGROUND Circulating tumor DNA (ctDNA) has emerged as a good candidate for tracking tumor dynamics in different cancer types, potentially avoiding repeated tumor biopsies. Many different genes can be mutated within a tumor, complicating procedures for tumor monitoring, even with highly sensitive next-generation sequencing (NGS) strategies. Droplet-based digital PCR (dPCR) is a highly sensitive and quantitative procedure, allowing detection of very low amounts of circulating tumor genetic material, but can be limited in the total number of target loci monitored. METHODS We analyzed hypermethylation of 3 genes, by use of droplet-based dPCR in different stages of colorectal cancer (CRC), to identify universal markers for tumor follow-up. RESULTS Hypermethylation of WIF1 (WNT inhibitory factor 1) and NPY (neuropeptide Y) genes was significantly higher in tumor tissue compared to normal tissue, independently of tumor stage. All tumor tissues appeared positive for one of the 2 markers. Methylated ctDNA (MetctDNA) was detected in 80% of metastatic CRC and 45% of localized CRC. For samples with detectable mutations in ctDNA, MetctDNA and mutant ctDNA (MutctDNA) fractions were correlated. During follow-up of different stage CRC patients, MetctDNA changes allowed monitoring of tumor evolution. CONCLUSIONS These results indicate that MetctDNA could be used as a universal surrogate marker for tumor follow-up in CRC patients, and monitoring MetctDNA by droplet-based dPCR could avoid the need for monitoring mutations.

Tumor angiogenesis promotes tumor growth and metastasis. Anti-angiogenic therapy in combination with chemotherapy is used for the treatment of metastatic cancers, including breast cancer but therapeutic benefits are limited. Mobilization and accumulation of myeloid-derived suppressor cells (MDSC) during tumor progression and therapy have been implicated in metastasis formation and resistance to antiangiogenic treatments. Here, we used the 4T1 orthotopic syngenic mouse model of mammary adenocarcinoma to investigate the effect of VEGF/VEGFR-2 axis inhibition on lung metastasis, MDSC and regulatory T cells (Tregs). We show that treatment with the anti-VEGFR-2 blocking antibody DC101 inhibits primary tumor growth, angiogenesis and lung metastasis. DC101 treatment had no effect on MDSC mobilization, but partially attenuated the inhibitory effect of mMDSC on T cell proliferation and decreased the frequency of Tregs in primary tumors and lung metastases. Strikingly, DC101 treatment induced the expression of the immune-suppressive molecule arginase I in mMDSC. Treatment with the arginase inhibitor N v -hydroxy-nor-Arginine (Nor-NOHA) reduced the inhibitory effect of MDSC on T cell proliferation and inhibited number and size of lung metastasis but had little or no additional effects in combination with DC101.In conclusion, DC101 treatment suppresses 4T1 tumor growth and metastasis, partially reverses the inhibitory effect of mMDSC on T cell proliferation, decreases Tregs in tumors and increases arginase I expression in mMDSC. Arginase inhibition suppresses lung metastasis independently of DC101 effects. These observations contribute to the further characterization of the immunomodulatory effect of anti-VEGF/VEGFR2 therapy and provide a rationale to pursue arginase inhibition as potential anti-metastatic therapy.

The Fas receptor ligand FasL regulates immune cell levels by inducing apoptosis of Fas receptor-positive cells. Here, we studied the impact of host FasL on tumor development in mice. Genetically targeting FasL in na€ ve mice increased myeloid cell populations, but, in marked contrast, it reduced the levels of myeloid-derived suppressor cells (MDSC) in mice bearing Lewis lung carcinoma tumors. Analysis of the MDSC subset distribution revealed that FasL deficiency skewed cell populations toward the M-MDSC subset, which displays a highly immunosuppressive activity. Furthermore, tumor-bearing mice that were FasL-deficient displayed an enhanced proportion of tumorassociated macrophages and regulatory T cells. Overall, the immunosuppressive environment produced by FasL targeting correlated with reduced survival of tumor-bearing mice. These results disclose a new role for FasL in modulating immunosuppressive cells. Cancer Res; 75(20); 4292-301. Ó2015 AACR.