Purpose Mutations in the estrogen receptor-alpha (ER) gene, ESR1, have been identified in breast cancer metastases after progression on endocrine therapies. Due to limitations of metastatic biopsies, the reported frequency of ESR1 mutations may be underestimated. Here, we show a high frequency of ESR1 mutations using circulating plasma tumor DNA (ptDNA) from metastatic breast cancer patients. Experimental Design We retrospectively obtained plasma samples from eight patients with known ESR1 mutations and three patients with wild type ESR1 identified by next generation sequencing (NGS) of biopsied metastatic tissues. Three common ESR1 mutations were queried for using droplet digital polymerase chain reaction (ddPCR). In a prospective cohort, metastatic tissue and plasma were collected contemporaneously from eight ER-positive and four ER-negative patients. Tissue biopsies were sequenced by NGS and ptDNA ESR1 mutations were analyzed by ddPCR. Results In the retrospective cohort, all corresponding mutations were detected in ptDNA, with two patients harboring additional ESR1 mutations not present in their metastatic tissues. In the prospective cohort, three ER-positive patients did not have adequate tissue for NGS, and no ESR1 mutations were identified in tissue biopsies from the other nine patients. In contrast, ddPCR detected seven ptDNA ESR1 mutations in six of twelve patients (50%). Conclusions We show that ESR1 mutations can occur at a high frequency and suggest that blood can be used to identify additional mutations not found by sequencing of a single metastatic lesion.

Ki-67 expression is correlated with cell proliferation and is a prognostic marker for various cancers; however, its function is unknown. Here we demonstrate that genetic disruption of Ki-67 in human epithelial breast and colon cancer cells depletes the cancer stem cell niche. Ki-67 null cells had a proliferative disadvantage compared to wildtype controls in colony formation assays and displayed increased sensitivity to various chemotherapies. Ki-67 null cancer cells showed decreased and delayed tumor formation in xenograft assays, which was associated with a reduction in cancer stem cell markers. Immunohistochemical analyses of human breast cancers revealed that Ki-67 expression is maintained at equivalent or greater levels in metastatic sites of disease compared to matched primary tumors, suggesting that maintenance of Ki-67 expression is associated with metastatic/clonogenic potential. These results elucidate Ki-67's role in maintaining the cancer stem cell niche, which has potential diagnostic and therapeutic implications for human malignancies.

Bone marrow transplantation (BMT) represents a cure for nonmalignant hematological disorders. However, compared with the stringent conditioning regimens used when performing BMT to treat hematological malignancies, the reduced intensity conditioning regimen used in the context of nonmalignant hematological disorders leads to substantially higher rates of BMT rejection, presumably due to an intact immune system. The relevant patient population typically receives transfusion support, often including platelets, and the frequency of BMT rejection correlates with the frequency of transfusion. Here, we demonstrate that immunity to transfused platelets contributes to subsequent BMT rejection in mice, even when the BMT donor and recipient are MHC matched. We used MHC-matched bone marrow because, although immunity to transfused platelets is best characterized in relation to HLA-specific antibodies, such antibodies are unlikely to play a role in clinical BMT rejection that is HLA matched. However, bone marrow is not matched in the clinic for minor histocompatibility antigens, such as those carried by platelets, and we report that transfusion of minor histocompatibility antigen-mismatched platelets induced subsequent BMT rejection. These findings indicate previously unappreciated sequelae of immunity to platelets in the context of transplantation and suggest that strategies to account for minor histocompatibility mismatching may help to reduce the chance of BMT rejection in human patients.