Phenotypic Analysis of Human Lymph Nodes in Subjects With New-Onset Type 1 Diabetes and Healthy Individuals by Flow Cytometry.

Background: Ultrasound guided sampling of human lymph node (LN) combined with advanced flow cytometry allows phenotypic analysis of multiple immune cell subsets. These may provide insights into immune processes and responses to immunotherapies not apparent from analysis of the blood. Methods: Ultrasound guided inguinal LN samples were obtained by both fine needle aspiration (FNA) and core needle biopsy in 10 adults within 8 weeks of diagnosis of type 1 diabetes (T1D) and 12 age-matched healthy controls at two study centers. Peripheral blood mononuclear cells (PBMC) were obtained on the same occasion. Samples were transported same day to the central laboratory and analyzed by multicolour flow cytometry. Results: LN sampling was well-tolerated and yielded sufficient cells for analysis in 95% of cases. We confirmed the segregation of CD69+ cells into LN and the predominance of CD8+ Temra cells in blood previously reported. In addition, we demonstrated clear enrichment of CD8+ naïve, FOXP3+ Treg, class-switched B cells, CD56bright NK cells and plasmacytoid dendritic cells (DC) in LNs as well as CD4+ T cells of the Th2 phenotype and those expressing Helios and Ki67. Conventional NK cells were virtually absent from LNs as were Th22 and Th1Th17 cells. Paired correlation analysis of blood and LN in the same individuals indicated that for many cell subsets, especially those associated with activation: such as CD25+ and proliferating (Ki67+) T cells, activated follicular helper T cells and class-switched B cells, levels in the LN compartment could not be predicted by analysis of blood. We also observed an increase in Th1-like Treg and less proliferating (Ki67+) CD4+ T cells in LN from T1D compared to control LNs, changes which were not reflected in the blood. Conclusions: LN sampling in humans is well-tolerated. We provide the first detailed "roadmap" comparing immune subsets in LN vs. blood emphasizing a role for differentiated effector T cells in the blood and T cell regulation, B cell activation and memory in the LN. For many subsets, frequencies in blood, did not correlate with LN, suggesting that LN sampling would be valuable for monitoring immuno-therapies where these subsets may be impacted.

Acquired Resistance of ER-Positive Breast Cancer to Endocrine Treatment Confers an Adaptive Sensitivity to TRAIL through Posttranslational Downregulation of c-FLIP.

Purpose: One third of ER-positive breast cancer patients who initially respond to endocrine therapy become resistant to treatment. Such treatment failure is associated with poor prognosis and remains an area of unmet clinical need. Here, we identify a specific posttranslational modification that occurs during endocrine resistance and which results in tumor susceptibility to the apoptosis-inducer TRAIL. This potentially offers a novel stratified approach to targeting endocrine-resistant breast cancer.Experimental Design: Cell line and primary-derived xenograft models of endocrine resistance were investigated for susceptibility to TRAIL. Tumor viability, cancer stem cell (CSC) viability (tumorspheres), tumor growth kinetics, and metastatic burden were assessed. Western blots for the TRAIL-pathway inhibitor, c-FLIP, and upstream regulators were performed. Results were confirmed in primary culture of 26 endocrine-resistant and endocrine-naïve breast tumors.Results: Breast cancer cell lines with acquired resistance to tamoxifen (TAMR) or faslodex were more sensitive to TRAIL than their endocrine-sensitive controls. Moreover, TRAIL eliminated CSC-like activity in TAMR cells, resulting in prolonged remission of xenografts in vivo In primary culture, TRAIL significantly depleted CSCs in 85% endocrine-resistant, compared with 8% endocrine-naïve, tumors, whereas systemic administration of TRAIL in endocrine-resistant patient-derived xenografts reduced tumor growth, CSC-like activity, and metastases. Acquired TRAIL sensitivity correlated with a reduction in intracellular levels of c-FLIP, and an increase in Jnk-mediated phosphorylation of E3-ligase, ITCH, which degrades c-FLIP.Conclusions: These results identify a novel mechanism of acquired vulnerability to an extrinsic cell death stimulus, in endocrine-resistant breast cancers, which has both therapeutic and prognostic potential. Clin Cancer Res; 24(10); 2452-63. ©2018 AACR.

Giant pseudoangiomatous stromal hyperplasia presenting in the breast of a prepubertal child.

Large benign lesions of the breasts are rare in children. We present a case of a 35 cm mass, weighing 2.7 kg in a 13-year-old girl with small developing breasts. Despite the enormity of the lesion, the patient managed to keep it concealed from her parents for 8 months. While initially suspicious of sarcoma a diagnosis of pseudoangiomatous stromal hyperplasia was suggested radiologically and confirmed histologically. Excision with reduction mammoplasty was performed, care taken not to disrupt the remaining breast tissue to facilitate future breast development. 18 months on, the cosmetic appearance of the breasts is good, with healthy underlying breast tissue developing. To the best of our knowledge this case is the largest documented breast tumour of this type in a patient of this age and illustrates the challenge of treating such tumours in the developing breast.

Fine-Needle Aspiration Biopsy of the Lymph Node: A Novel Tool for the Monitoring of Immune Responses after Skin Antigen Delivery.

Assessment of immune responses in lymph nodes (LNs) is routine in animals, but rarely done in humans. We have applied minimally invasive ultrasound-guided fine-needle aspiration of the LN to a before-and-after study of the immune response to intradermally delivered Ag in healthy volunteers (n = 25). By comparison with PBMCs from the same individual, LN cells (LNCs) were characterized by reduced numbers of effector memory cells, especially CD8(+) TEMRA cells (3.37 ± 1.93 in LNCs versus 22.53 ± 7.65 in PBMCs; p = 0.01) and a marked increased in CD69 expression (27.67 ± 7.49 versus 3.49 ± 2.62%, LNCs and PBMCs, respectively; p < 0.0001). At baseline, there was a striking absence of IFN-γ ELISPOT responses to recall Ags (purified protein derivative, Tetanus toxoid, or flu/EBV/CMV viral mix) in LN, despite strong responses in the peripheral blood. However, 48 h after tuberculin purified protein derivative administration in the ipsilateral forearm resulting in a positive skin reaction, a clear increase in IFN-γ ELISPOT counts was seen in the draining LN but not in PBMCs. This response was lost by 5 d. These data suggest that the low levels of effector memory cells in the LN may explain the low background of baseline ELISPOT responses in LNs as compared with PBMCs, and the appearance of a response after 48 h is likely to represent migration of effector memory cells from the skin to the LN. Hence, it appears that the combination of intradermal Ag administration and draining LN sampling can be used as a sensitive method to probe the effector memory T cell repertoire in the skin.