Molecular signatures define alopecia areata subtypes and transcriptional biomarkers

AbstractAlopecia areata (AA) is an autoimmune disease typified by nonscarring hair loss with a variable clinical course. In this study, we conducted whole genome gene expression analysis of 96 human scalp skin biopsy specimens from AA or normal control subjects. Based on gene expression profiling, samples formed distinct clusters based on the presence or absence of disease as well as disease phenotype (patchy disease compared with alopecia totalis or universalis). Differential gene expression analysis allowed us to robustly demonstrate graded immune activity in samples of increasing phenotypic severity and generate a quantitative gene expression scoring system that classified samples based on interferon and cytotoxic T lymphocyte immune signatures critical for disease pathogenesis

HER2 testing in breast cancer: Opportunities and challenges

Human epidermal growth factor receptor 2 (HER2) is overexpressed in 15-25% of breast cancers, usually as a result of HER2 gene amplification. Positive HER2 status is considered to be an adverse prognostic factor. Recognition of the role of HER2 in breast cancer growth has led to the development of anti-HER2 directed therapy, with the humanized monoclonal antibody trastuzumab (Herceptin (R)) having been approved for the therapy of HER2-positive metastatic breast cancer. Clinical studies have further suggested that HER2 status can provide important information regarding success or failure of certain hormonal therapies or chemotherapies. As a result of these developments, there has been increasing demand to perform HER2 testing on current and archived breast cancer specimens. This article reviews the molecular background of HER2 function, activation and inhibition as well as current opinions concerning its role in chemosensitivity and interaction with estrogen receptor biology. The different tissue-based assays used to detect HER2 amplification and overexpression are discussed with respect to their advantages and disadvantages, when to test (at initial diagnosis or pre-treatment), where to test (locally or centralized) and the need for quality assurance to ensure accurate and valid testing results

Markedly Different Pathogenicity of Four Immunoglobulin G Isotype-Switch Variants of an Antierythrocyte Autoantibody Is Based on Their Capacity to Interact in Vivo with the Low-Affinity Fcγ Receptor III

Using three different Fcγ receptor (FcγR)-deficient mouse strains, we examined the induction of autoimmune hemolytic anemia by each of the four immunoglobulin (Ig)G isotype-switch variants of a 4C8 IgM antierythrocyte autoantibody and its relation to the contributions of the two FcγR, FcγRI, and FcγRIII, operative in the phagocytosis of opsonized particles. We found that the four IgG isotypes of this antibody displayed striking differences in pathogenicity, which were related to their respective capacity to interact in vivo with the two phagocytic FcγRs, defined as follows: IgG2a > IgG2b > IgG3/IgG1 for FcγRI, and IgG2a > IgG1 > IgG2b > IgG3 for FcγRIII. Accordingly, the IgG2a autoantibody exhibited the highest pathogenicity, ∼20–100-fold more potent than its IgG1 and IgG2b variants, respectively, while the IgG3 variant, which displays little interaction with these FcγRs, was not pathogenic at all. An unexpected critical role of the low-affinity FcγRIII was revealed by the use of two different IgG2a anti–red blood cell autoantibodies, which displayed a striking preferential utilization of FcγRIII, compared with the high-affinity FcγRI. This demonstration of the respective roles in vivo of four different IgG isotypes, and of two phagocytic FcγRs, in autoimmune hemolytic anemia highlights the major importance of the regulation of IgG isotype responses in autoantibody-mediated pathology and humoral immunity

A biophysical model of cell adhesion mediated by immunoadhesin drugs and antibodies

A promising direction in drug development is to exploit the ability of natural killer cells to kill antibody-labeled target cells. Monoclonal antibodies and drugs designed to elicit this effect typically bind cell-surface epitopes that are overexpressed on target cells but also present on other cells. Thus it is important to understand adhesion of cells by antibodies and similar molecules. We present an equilibrium model of such adhesion, incorporating heterogeneity in target cell epitope density and epitope immobility. We compare with experiments on the adhesion of Jurkat T cells to bilayers containing the relevant natural killer cell receptor, with adhesion mediated by the drug alefacept. We show that a model in which all target cell epitopes are mobile and available is inconsistent with the data, suggesting that more complex mechanisms are at work. We hypothesize that the immobile epitope fraction may change with cell adhesion, and we find that such a model is more consistent with the data. We also quantitatively describe the parameter space in which binding occurs. Our results point toward mechanisms relating epitope immobility to cell adhesion and offer insight into the activity of an important class of drugs.Comment: 13 pages, 5 figure

Serial Killing of Tumor Cells by Human Natural Killer Cells – Enhancement by Therapeutic Antibodies

BACKGROUND: Natural killer cells are an important component of the innate immune system. Anti-cancer therapies utilizing monoclonal antibodies also rely on the cytotoxicity of NK cells for their effectiveness. Here, we study the dynamics of NK cell cytotoxicity. METHODOLOGY/PRINCIPAL FINDINGS: We observe that IL-2 activated human NK cells can serially hit multiple targets. Using functional assays, we demonstrate that on an average, a single IL-2 activated NK cell can kill four target cells. Data using live video microscopy suggest that an individual NK cell can make serial contacts with multiple targets and majority of contacts lead to lysis of target cells. Serial killing is associated with a loss of Perforin and Granzyme B content. A large majority of NK cells survive serial killing, and IL-2 can replenish their granular stock and restore the diminished cytotoxicity of ‘exhausted’ NK cells. IL-2 and IL-15 are equally effective in enhancing the killing frequency of resting NK cells. Significantly, Rituximab, a therapeutic monoclonal antibody increases the killing frequency of both resting and IL-2 activated NK cells. CONCLUSION/SIGNIFICANCE: Our data suggest that NK cell-based therapies for overcoming tumors rely on their serial killing ability. Therefore, strategies augmenting the killing ability of NK cells can boost the immune system and enhance the effectiveness of monoclonal antibody-based therapies

Membrane transport proteins in human melanoma: associations with tumour aggressiveness and metastasis

BACKGROUND: Malignant melanoma, generally described as incurable, is notoriously refractory to chemotherapy. The mechanisms contributing to this have not yet been defined and the contributions of drug efflux pumps, implicated in chemo-resistance of many other cancer types, have not been extensively investigated in melanoma. METHODS: In this study, expression of multi-drug resistant (MDR1/P-gp and MRP-1) proteins was examined, by immunohistochemistry, in archival specimens from 134 melanoma patients. This included 92 primary tumours and 42 metastases. RESULTS: On assessing all specimens, MRP-1 and MDR1/P-gp expression was found to be common, with the majority (81%) of melanomas expressing at least one of these efflux pumps. Although there is significant association between expression of these pumps (P=0.007), MRP-1 was found to be the predominant (67% of cases) form detected. chi(2) analysis showed significant associations between expression of MRP-1 and/or MDR1/P-gp and the aggressive nature of this disease specifically increased Breslow's depth, Clark's level and spread to lymph nodes. This association with aggressiveness and spread is further supported by the observation that a significantly higher percentage of metastases, than primary tumours, express MRP-1 (91% vs 57%; P<0.0001) and MDR1/P-gp (74% vs 50%; P=0.010). CONCLUSION: The predominant expression of these pumps and, in particular, MRP-1 suggests that they may be important contributors to the inherent aggressive and resistant nature of malignant melanoma

Trastuzumab-DM1 causes tumour growth inhibition by mitotic catastrophe in trastuzumab-resistant breast cancer cells in vivo

Introduction Trastuzumab is widely used for the treatment of HER2-positive breast cancer. Despite encouraging clinical results, a significant fraction of patients are, or become, refractory to the drug. To overcome this, trastuzumab-DM1 (T-DM1), a newer, more potent drug has been introduced. We tested the efficacy and mechanisms of action of T-DM1 in nine HER2-positive breast cancer cell lines in vitro and in vivo. The nine cell lines studied included UACC-893, MDA-453 and JIMT-1, which are resistant to both trastuzumab and lapatinib. Methods AlamarBlue cell-proliferation assay was used to determine the growth response of breast cancer cell lines to trastuzumab and T-DM1 in vitro. Trastuzumab- and T-DM1-mediated antibody-dependent cellular cytotoxicity (ADCC) was analysed by measuring the lactate dehydrogenase released from the cancer cells as a result of ADCC activity of peripheral blood mononuclear cells. Severe Combined Immunodeficient (SCID) mice were inoculated with trastuzumab-resistant JIMT-1 cells to investigate the tumour inhibitory effect of T-DM1 in vivo. The xenograft samples were investigated using histology and immunohistochemistry. Results T-DM1 was strongly growth inhibitory on all investigated HER2-positive breast cancer cell lines in vitro. T-DM1 also evoked antibody-dependent cellular cytotoxicity (ADCC) similar to that of trastuzumab. Outgrowth of JIMT-1 xenograft tumours in SCID mice was significantly inhibited by T-DM1. Histologically, the cellular response to T-DM1 consisted of apoptosis and mitotic catastrophe, the latter evidenced by an increased number of cells with aberrant mitotic figures and giant multinucleated cells. Conclusions Our results suggest mitotic catastrophe as a previously undescribed mechanism of action of T-DM1. T-DM1 was found effective even on breast cancer cell lines with moderate HER2 expression levels and cross-resistance to trastuzumab and lapatinib (MDA-453 and JIMT-1).BioMed Central Open acces

hI-con1, a factor VII-IgGFc chimeric protein targeting tissue factor for immunotherapy of uterine serous papillary carcinoma

BACKGROUND: Uterine serous papillary adenocarcinoma (USPC) is a highly aggressive variant of endometrial cancer. Human immunoconjugate molecule (hI-con1) is an antibody-like molecule targeted against tissue factor (TF), composed of two human Factor VII (fVII) as the targeting domain, fused to human immunoglobulin (Ig) G1 Fc as an effector domain. We evaluated hI-con1 potential activity against primary chemotherapy-resistant USPC cell lines expressing different levels of TF. METHODS: A total of 16 formalin-fixed, paraffin-embedded USPC samples were evaluated by immunohistochemistry (IHC) for TF expression. Six primary USPC cell lines, half of which overexpress the epidermal growth factor type II (HER2/neu) receptor at 3\ufe levels, were assessed by flow cytometry and real-time PCR for TF expression. Sensitivity to hI-con1-dependent cell-mediated cytotoxicity (IDCC) was evaluated in 5-hour-chromium release assays. Finally, to investigate the effect of interleukin-2 (IL-2) on IDCC, 5-h 51Cr assays were also conducted in the presence of low doses of IL-2 (i.e., 50\u2013100 IU ml 1). RESULTS: Cytoplasmic and/or membrane TF expression was observed in all 16 (100%) USPC samples tested by IHC, but not in normal endometrium. High expression of TF was found in 50% (three out of six) of the USPC cell lines tested by real-time PCR and flow cytometry when compared with normal endometrial cells (NECs; Po0.001). Uterine serous papillary adenocarcinoma cell lines overexpressing TF, regardless of their high or low HER2/neu expression, were highly sensitive to IDCC (mean killing\ub1s.d., 65.6\ub13.7%, range 57.5\u201377.0%, Po0.001), although negligible cytotoxicity against USPC was seen in the absence of hI-con1 or in the presence of Rituximab control antibody. The addition of low doses of IL-2 further increased the cytotoxic effect induced by hI-con1 against chemotherapy-resistant USPC. CONCLUSION: hI-con1 induces strong cytotoxicity against primary chemotherapy-resistant USPC cell lines overexpressing TF. The hI-con1 may represent a novel therapeutic agent for the treatment of patients harbouring advanced, recurrent and/or metastatic USPC refractory to standard treatment modalities

Prognostic importance of survivin in breast cancer

Survivin is a member of the inhibitor of apoptosis (IAP) family, and is also involved in the regulation of cell division. Survivin is widely expressed in foetal tissues and in human cancers, but generally not in normal adult tissue. This study examined the expression of surviving protein in a series of 293 cases of invasive primary breast carcinoma. Survivin immunoreactivity was assessed using two different polyclonal antibodies, and evaluated semiquantitatively according to the percentage of cells demonstrating distinct nuclear and/or diffuse cytoplasmic staining. Overall, 60% of tumours were positive for survivin: 31% demonstrated nuclear staining only, 13% cytoplasmic only, and 16% of tumour cells demonstrated both nuclear and cytoplasmic staining. Statistical analysis revealed that survivin expression was independent of patient's age, tumour size, histological grade, nodal status, and oestrogen receptor status. In multivariate analysis, nuclear survivin expression was a significant independent prognostic indicator of favourable outcome both in relapse-free and overall survival (P<0.001 and P=0.01, respectively). In conclusion, our results show that survivin is frequently overexpressed in primary breast cancer. Nuclear expression is most common and is an independent prognostic indicator of good prognosis

NK-cell and T-cell functions in patients with breast cancer: effects of surgery and adjuvant chemo- and radiotherapy

Breast cancer is globally the most common malignancy in women. Her2-targeted monoclonal antibodies are established treatment modalities, and vaccines are in late-stage clinical testing in patients with breast cancer and known to promote tumour-killing through mechanisms like antibody-dependent cellular cytotoxicity. It is therefore increasingly important to study immunological consequences of conventional treatment strategies. In this study, functional tests and four-colour flow cytometry were used to detect natural killer (NK)-cell functions and receptors as well as T-cell signal transduction molecules and intracellular cytokines in preoperative breast cancer patients, and patients who had received adjuvant radiotherapy or adjuvant combined chemo-radiotherapy as well as in age-matched healthy controls. The absolute number of NK cells, the density of NK receptors as well as in vitro quantitation of functional NK cytotoxicity were significantly higher in preoperative patients than the post-treatments group and controls. A similar pattern was seen with regard to T-cell signalling molecules, and preoperative patients produced significantly higher amounts of cytokines in NK and T cells compared to other groups. The results indicate that functions of NK and T cells are well preserved before surgery but decrease following adjuvant therapy, which may speak in favour of early rather than late use of immunotherapeutic agents such as trastuzumab that may depend on intact immune effector functions