Circulating Tumor DNA and Late Recurrence in High-Risk Hormone Receptor-Positive, Human Epidermal Growth Factor Receptor 2-Negative Breast Cancer.

PURPOSE: To examine the prevalence and dynamics of circulating tumor DNA (ctDNA) and its association with metastatic recurrence in patients with high-risk early-stage hormone receptor-positive breast cancer (HR+ BC) more than 5 years from diagnosis. METHODS: We enrolled 103 patients with high-risk stage II-III HR+ BC diagnosed more than 5 years prior without clinical evidence of recurrence. We performed whole-exome sequencing (WES) on primary tumor tissue to identify somatic mutations tracked via a personalized, tumor-informed ctDNA test to detect minimal residual disease (MRD). We collected plasma at the time of consent and at routine visits every 6-12 months. Patients were followed for clinical recurrence. RESULTS: In total, 85 of 103 patients had sufficient tumor tissue; of them, 83 of 85 (97.6%) patients had successful whole-exome sequencing. Personalized ctDNA assays were designed targeting a median of 36 variants to test 219 plasma samples. The median time from diagnosis to first sample was 8.4 years. The median follow-up was 10.4 years from diagnosis and 2.0 years from first sample. The median number of plasma samples per patient was two. Eight patients (10%) had positive MRD testing at any time point. Six patients (7.2%) developed distant metastatic recurrence, all of whom were MRD-positive before overt clinical recurrence, with median ctDNA lead time of 12.4 months. MRD was not identified in one patient (1.2%) with local recurrence. Two of eight MRD-positive patients had not had clinical recurrence at last follow-up. CONCLUSION: In this prospective study, in patients with high-risk HR+ BC in the late adjuvant setting, ctDNA was identified a median of 1 year before all cases of distant metastasis. Future studies will determine if ctDNA-guided intervention in patients with HR+ BC can alter clinical outcomes.

APOE ε 4 allele and CSF APOE on cognition in HIV-infected subjects.

The significance of the cerebrospinal fluid (CSF) Apolipoprotein E (APOE) level and whether it might have differential effects on brain function due to the presence of APOE ε 4 allele(s) in HIV-infected patients are unknown. However, APOE ε 4 allele has been associated with greater incidence of HIV-associated dementia and accelerated progression of HIV infection. Here, we show further evidence for the role of APOE ε 4 in promoting cognitive impairment. We measured the APOE levels in the CSF of HIV-infected individuals. HIV+ subjects showed lower CSF APOE proteins than SN controls (-19%, p= 0.03). While SN subjects with or without ε 4 allele showed no difference in CSF APOE levels, ε 4+ HIV+ subjects had similar levels to the SN subjects but higher levels than ε 4- HIV+ subjects (+34%, p= 0.01). Furthermore, while HIV+ subjects with ε 2 or ε 3 allele(s) showed a positive relationship between their CSF APOE levels and cognitive performance on the speed of processing domain (r= +0.35, p= 0.05), ε 4+ HIV+ subjects, in contrast, exhibited a negative relationship such that those with higher levels of CSF APOE(4) performed worse on the HIV Dementia Scale (r= -0.61, p= 0.02), had lower Global Cognitive Scores (r= -0.57, p= 0.03), and had poorer performance on tests involving learning (ε 4 allele x [APOE] interaction, p = 0.01). Our findings also suggest that the relatively higher levels of CSF APOE in ε 4+ HIV+ (having primarily APOE4 isoforms) may negatively impact the brain and lead to poorer cognitive outcomes, while those individuals without the ε 4 allele (with primarily APOE2 or APOE3 isoforms) may show compensatory responses that lead to better cognitive performance.

HLA-G expression defines a novel regulatory T-cell subset present in human peripheral blood and sites of inflammation.

Regulatory T cells can inhibit harmful immunopathologic responses directed against self and foreign antigens and play a major role in controlling autoimmunity. Here we have identified and characterized a subpopulation of CD4 and CD8 T cells in human peripheral blood expressing the immune tolerizing molecule HLA-G. HLA-G-expressing T cells are hypoproliferative, are CD25- and FOXP3-negative, and exhibit potent suppressive properties that are partially mediated by HLA-G. HLA-G-positive (HLA-G(pos)) T cells are found at low percentages among CD4 and CD8 single-positive thymocytes, suggesting a thymic origin. The presence of HLA-G(pos) T cells at sites of inflammation such as inflamed skeletal muscle in myositis or the cerebrospinal fluid of patients with acute neuroinflammatory disorders suggests an important function in modulating parenchymal inflammatory responses in vivo.

Expression of the immune-tolerogenic major histocompatibility molecule HLA-G in multiple sclerosis: implications for CNS immunity.

HLA-G is a non-classical major histocompatibility complex (MHC) class I antigen with highly limited tissue distribution under non-pathological conditions. Although capable of acting as a peptide-presenting molecule, its strong immune-inhibitory properties identify HLA-G as a mediator of immune tolerance with specific relevance at immune-privileged sites such as trophoblast or thymus. To assess the role of HLA-G in CNS immunity, we investigated its expression in brain specimens from patients with multiple sclerosis (n = 11), meningitis (n = 2) and Alzheimer's disease (n = 2) and non-pathological CNS controls (n = 6). Furthermore, cultured human microglial cells and CSF of patients with multiple sclerosis and controls were assessed. Furthermore, CSF from MS patients and controls, as well as cultured human microglial cells were assessed. Using several HLA-G specific mAb and immunohistochemistry, HLA-G protein was found strongly expressed in brain specimens from patients with multiple sclerosis while it was rarely detectable in the non-pathological control specimens. In multiple sclerosis brain specimens, HLA-G immunoreactivity was observed in acute plaques, in chronic active plaques, in perilesional areas as well as in normal appearing white matter. In all areas microglial cells, macrophages, and in part endothelial cells were identified as the primary cellular source of expression. HLA-G was also found in other disease entities (meningitis, Alzheimer's specimens) where expression correlated to activation and MHC class II expression on microglial cells. Importantly, ILT2, a receptor for HLA-G, was also found in multiple sclerosis brain specimens thus emphasizing the relevance of this inhibitory pathway in vivo. HLA-G mRNA and protein expression and regulation could also be corroborated on cultured human microglial cells in vitro. Further, expression of HLA-G in the CSF of multiple sclerosis patients and controls was analysed by flow cytometry and ELISA. Monocytes represented the main source of cellular HLA-G expression in the CSF. Corresponding to the observations with the tissue specimens, CSF mean levels of soluble HLA-G were significantly higher in multiple sclerosis than in non-inflammatory controls (171 +/- 31 versus 39 +/- 10 U/ml; P = 0.0001). The demonstration of HLA-G and its receptor ILT2 on CNS cells and in areas of microglia activation implicate HLA-G as a contributor to the fundamental mechanisms regulating immune reactivity in the CNS. This pathway may act as an inhibitory feedback aimed to downregulate the deleterious effects of T-cell infiltration in neuroinflammation.

Systemic NKG2D down-regulation impairs NK and CD8 T cell responses in vivo.

The immunoreceptor NKG2D stimulates activation of cytotoxic lymphocytes upon engagement with MHC class I-related NKG2D ligands of which at least some are expressed inducibly upon exposure to carcinogens, cell stress, or viruses. In this study, we investigated consequences of a persistent NKG2D ligand expression in vivo by using transgenic mice expressing MHC class I chain-related protein A (MICA) under control of the H2-K(b) promoter. Although MICA functions as a potent activating ligand of mouse NKG2D, H2-K(b)-MICA mice appear healthy without aberrations in lymphocyte subsets. However, NKG2D-mediated cytotoxicity of H2-K(b)-MICA NK cells is severely impaired in vitro and in vivo. This deficiency concurs with a pronounced down-regulation of surface NKG2D that is also seen on activated CD8 T cells. As a consequence, H2-K(b)-MICA mice fail to reject MICA-expressing tumors and to mount normal CD8 T cell responses upon Listeria infection emphasizing the importance of NKG2D in immunity against tumors and intracellular infectious agents.