Background:Malignant transformation requires the interaction of cancer cells with their microenvironment, including infiltrating leukocytes. However, somatic mutational studies have focused on alterations in cancer cells, assuming that the microenvironment is genetically normal. Because we hypothesized that this might not be a valid assumption, we performed exome sequencing and targeted sequencing to investigate for the presence of pathogenic mutations in tumor-associated leukocytes in breast cancers.Methods:We used targeted sequencing and exome sequencing to evaluate the presence of mutations in sorted tumor-infiltrating CD45-positive cells from primary untreated breast cancers. We used high-depth sequencing to determine the presence/absence of the mutations we identified in breast cancer-infiltrating leukocytes in purified tumor cells and in circulating blood cells.Results:Capture-based sequencing of 15 paired tumor-infiltrating leukocytes and matched germline DNA identified variants in known cancer genes in all 15 primary breast cancer patients in our cohort. We validated the presence of mutations identified by targeted sequencing in infiltrating leukocytes through orthogonal exome sequencing. Ten patients harbored alterations previously reported as somatically acquired variants, including in known leukemia genes (DNTM3A, TET2, and BCOR). One of the mutations observed in the tumor-infiltrating leukocytes was also detected in the circulating leukocytes of the same patients at a lower allele frequency than observed in the tumor-infiltrating cells.Conclusions:Here we show that somatic mutations, including mutations in known cancer genes, are present in the leukocytes infiltrating a subset of primary breast cancers. This observation allows for the possibility that the cancer cells interact with mutant infiltrating leukocytes, which has many potential clinical implications.
SummaryTriple negative breast cancer (TNBC) is an aggressive type of cancer that is known to be resistant to radiotherapy (RT). Evidence is accumulating that is indicative of the plasticity of TNBC, where one cancer subtype switches to another in response to various treatments, including RT. In this study we aim to overcome tumor resistance by designing TNBC-sensitizing targeted therapies that exploit the plasticity occurring due to radiation exposure. Using single cell analysis of molecular changes occurring in irradiated TNBC tumors, we identified two initially undetected distinct subpopulations, represented by overexpressed Her2 and cMet, expanding post-RT and persisting in surviving tumors. Using murine cancer models and patient-derived TNBC tumors, we showed that only simultaneous targeting of Her2 and cMet was successful in sensitizing TNBC to RT and preventing its regrowth. The strategy presented herein holds the potential to be broadly applicable in clinical use.HighlightsSensitization of TNBC to radiotherapy (RT) is a clinically unmet needSingle cell strategy creates a precise map of subpopulations expanding post-RTEvolution of intra-tumor heterogeneity is turned into a therapeutic advantageSimultaneous targeting of expanding subpopulations sensitizes TNBC to radiotherapy
Primary tumors have been shown to prepare distal organs for later colonization of metastatic cells by stimulating organ-specific infiltration of bone marrow derived cells. We have recently demonstrated that neutrophils are mobilized by the primary tumor and accumulate in the lung prior to the arrival of metastatic cells in mouse models of breast cancer. Tumor-entrained neutrophils (TENs) inhibit metastatic seeding in the lungs by generating H2O2 and tumor secreted CCL2 is both required and sufficient for optimal anti-metastatic entrainment of G-CSF-mobilized neutrophils. TENs are present in the peripheral blood of breast cancer patients prior to surgical resection but not in healthy individuals. Thus, while tumor-secreted factors contribute to tumor progression at the primary site, they concomitantly induce a neutrophil-mediated inhibitory process at the metastatic site. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr MS1-2.
e22148 Background: Recent research in murine breast cancer models demonstrates that primary breast tumors can mobilize select neutrophils, which have the unique capacity to inhibit metastatic seeding in the lung through cell-kill mechanisms. (Granot Z et al. Cancer Cell. 2010). In both breast cancer patients and healthy women, we investigated whether neutrophils were cytotoxic to breast cancer cell lines. We also assessed whether serum chemokines were associated with neutrophil cytotoxicity. Methods: Neutrophils were purified from the blood of 59 randomly selected newly diagnosed pre-operative breast cancer patients without evidence of metastatic disease, 50 metastatic breast cancer patients, 15 DCIS patients, and 45 healthy female volunteers with no history of cancer. Cytotoxicity was evaluated by incubating neutrophils with luciferase labeled MDA-MB-231 cells. Luciferase activity was measured as a reflection of % cytotoxicity. Serum was also isolated from breast cancer patients and healthy volunteers. Millipore Milliplex Human Cytokine Plex Kit was used to assess the concentration of 42 different chemokines in each of the serum samples. Results: In comparison to healthy volunteers whose mean neutrophil cytotoxicity to MDA-MB-231 cells was 6.5%, pre-operative breast cancer patients demonstrated a mean neutrophil cytotoxicity of 13.4%, p<0.0001, metastatic patients mean neutrophil cytotoxicity was 19.4%, p <0.003 and DCIS patients had a mean neutrophil cytotoxicity of 11.7%. p <0.01. By Kruskal-Wallist test, the relative concentrations of three chemokines, Il.1a, MCP.1 and TNF-a, were found to have a statistically significant association with neutrophil cytotoxicity (p-value: 0.005, 0.002, 0.0005 respectively). Conclusions: Our work demonstrates the cytotoxic role of select neutrophils in the peripheral blood of breast cancer patients as contrasted with neutrophils from healthy women. We further demonstrate that select chemokines appear to be correlated with neutrophil cytotoxicity, in varying concentrations. Further studies are needed to evaluate the prognostic and therapeutic role of cytotoxic neutrophils as well as the role of chemokines in neutrophil cytotoxicity.
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