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We have studied responses in thymoma patients to interferon-alpha and to the acetylcholine receptor (AChR) in early-onset myasthenia gravis (EOMG), seeking clues to autoimmunizing mechanisms. Our new evidence implicates a two-step process: (step 1) professional antigen-presenting cells and thymic epithelial cells prime AChR-specific T cells; then (step 2) thymic myoid cells subsequently provoke germinal center formation in EOMG. Our unifying hypothesis proposes that AChR epitopes expressed by neoplastic or hyperplastic thymic epithelial cells aberrantly prime helper T cells, whether generated locally or infiltrating from the circulation. These helper T cells then induce antibody responses against linear epitopes that cross-react with whole AChR and attack myoid cells in the EOMG thymus. The resulting antigen-antibody complexes and the recruitment of professional antigen-presenting cells increase the exposure of thymic cells to the infiltrates and provoke local germinal center formation and determinant spreading. Both these and the consequently enhanced heterogeneity and pathogenicity of the autoantibodies should be minimized by early thymectomy.

// Wing-yin Leung 1 , Ioannis Roxanis 2 , Helen Sheldon 1 , Francesca M. Buffa 1 , Ji-Liang Li 1 , Adrian L. Harris 1 and Anthony Kong 1,3 1 Department of Oncology, Molecular Oncology Laboratories, The Weatherall Institute of Molecular Medicine, University of Oxford, United Kingdom 2 Department of Cellular Pathology, Oxford University Hospitals and Oxford Biomedical Research Centre, Oxford, United Kingdom 3 New address: School of Cancer Sciences, University of Birmingham, Birmingham, United Kingdom Correspondence: Anthony Kong, email: // Adrian L. Harris, email: // Keywords : lapatinib, pertuzumab, resistance, NRG1, HER2 Received : November 13, 2014 Accepted : January 02, 2015 Published : January 21, 2015 Abstract Acquired resistance to lapatinib, an inhibitor of EGFR and HER2 kinases, is common. We found that reactivation of EGFR, HER2 and HER3 occurred within 24 hours of lapatinib treatment after their initial dephosphorylation. This was associated with increased expression of NRG1 in cells treated with lapatinib. Exogenous NRG1 partially rescued breast cancer cells from growth inhibition by lapatinib. In addition, both parental and lapatinib-resistant breast cancer cells were sensitive to SGP1, which inhibits binding of NRG1 and other HER3 ligands. Addition of pertuzumab to lapatinib further inhibited NRG1-induced signalling, which was not fully inhibited by either drug alone. In animal model, a combination of pertuzumab to lapatinib induced a greater tumor regression than either lapatinib or pertuzumab monotherapy. This novel combination treatment may provide a promising strategy in clinical HER2-targeted therapy and may inhibit a subset of lapatinib-resistant breast cancer, although the group of patients that will respond to this therapy requires further stratification.

// Katharina Feldinger 1 , Daniele Generali 3 , Gabriela Kramer-Marek 4,8 , Merel Gijsen 1 , Tzi Bun Ng 5 , Jack Ho Wong 5 , Carla Strina 3 , Mariarosa Cappelletti 3 , Daniele Andreis 3 , Ji-Liang Li 2 , Esther Bridges 2 , Helen Turley 2 , Russell Leek 2 , Ioannis Roxanis 6 , Jacek Capala 4 , Gillian Murphy 7 , Adrian L. Harris 2 and Anthony Kong 1 1 Human Epidermal Growth Factor Group, Department of Oncology, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK 2 Growth Factor Group, Department of Oncology, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK 3 U.O. Multidisciplinare di Patologia Mammaria, U.S Terapia Molecolare e Farmacogenomica, A.O. Instituti Ospitalieri di Cremona, Cremona, Italy 4 National Institutes of Health, Radiation Oncology Branch, Bethesda MD, US 5 School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Tai Po Road, Sha Tin, Hong Kong 6 Department of Cellular Pathology, Oxford University Hospitals and Oxford Biomedical Research Centre, Oxford, UK 7 Cancer Research UK Cambridge Institute, Li Ka Shing Centre, Robinson Way, Cambridge, UK 8 Institute of Cancer Research, Division of Radiotherapy and Imaging, Belmont, Sutton, Surrey, UK (New address) Correspondence: Anthony Kong, email: // Keywords : Trastuzumab, resistance, ADAM10, HER2, survival Received : March 8, 2014 Accepted : May 7, 2014 Published : May 8, 2014 Abstract Trastuzumab prolongs survival in HER2 positive breast cancer patients. However, resistance remains a challenge. We have previously shown that ADAM17 plays a key role in maintaining HER2 phosphorylation during trastuzumab treatment. Beside ADAM17, ADAM10 is the other well characterized ADAM protease responsible for HER ligand shedding. Therefore, we studied the role of ADAM10 in relation to trastuzumab treatment and resistance in HER2 positive breast cancer. ADAM10 expression was assessed in HER2 positive breast cancer cell lines and xenograft mice treated with trastuzumab. Trastuzumab treatment increased ADAM10 levels in HER2 positive breast cancer cells (p≤0.001 in BT474; p≤0.01 in SKBR3) and in vivo (p≤0.0001) compared to control, correlating with a decrease in PKB phosphorylation. ADAM10 inhibition or knockdown enhanced trastuzumab response in naive and trastuzumab resistant breast cancer cells. Trastuzumab monotherapy upregulated ADAM10 (p≤0.05); and higher pre-treatment ADAM10 levels correlated with decreased clinical response (p≤0.05) at day 21 in HER2 positive breast cancer patients undergoing a trastuzumab treatment window study. Higher ADAM10 levels correlated with poorer relapse-free survival (p≤0.01) in a cohort of HER2 positive breast cancer patients. Our studies implicate a role of ADAM10 in acquired resistance to trastuzumab and establish ADAM10 as a therapeutic target and a potential biomarker for HER2 positive breast cancer patients.

UNLABELLED: Currently, an alteration in the gross volume of a tumor is used to assess its response to trastuzumab; however, this approach provides only a late indication of response. Tissue-sample ex vivo assays are potentially valuable, but their procurement through biopsies is invasive and might be biased by tumor heterogeneity. We studied the feasibility of using PET to quantify changes in ErbB2 (HER2/neu) expression and to predict the response to trastuzumab in BT474 breast cancer xenografts with N-[2-(4-(18)F-fluorobenzamido)ethyl]maleimide ((18)F-FBEM)-HER(2:342) Affibody. METHODS: Mice bearing BT474 tumors were given trastuzumab (50 mg/kg loading dose, 25 mg/kg maintenance dose, administered intraperitoneally twice a week) or saline (control) for a total of 5 doses. Tumor size was monitored twice a week. Animals were scanned before the treatment, at 48 h, and 2 wk after the beginning of therapy. After the final scan, PET results were correlated with tumor response and immunohistochemical assessment of ErbB2 level, as well as with vasculature in the treated tumors. RESULTS: Analysis of PET images indicated that tracer uptake was significantly reduced after 1 dose of trastuzumab, compared with baseline, suggesting applicability as an early indicator of changes in ErbB2 expression. After 5 doses of trastuzumab, the overall decrease in (18)F-FBEM-HER(2:342) Affibody uptake also correlated with tumor response and downregulation of ErbB2 expression by immunohistochemical assessment. However, individual animals had different responses. There was a correlation between bigger PET changes and a higher vessel count in the tumors, suggesting that an increased number of vessels could lead to better trastuzumab delivery. We confirmed that the difference in average vessel count in the tumors was not related to the size of the tumors and therefore was not due to the selection of more vascular tumors. This finding is consistent with previous findings demonstrating that the number of vessels in a tumor could be a useful prognostic marker for treatment response. CONCLUSION: Our data suggest that Affibody-based PET can noninvasively provide specific information on changes in receptor expression and could be a valuable strategy for predicting tumor response to trastuzumab.

Malignant melanomas have a high metastatic potential. Although the depth of tumour invasion is the single most important histological prognostic factor, in clinical practice this correlation is frequently challenged. In this study, we assessed the cohesion of malignant melanocytes in the dermal component of all primary melanomas in vertical growth phase with tumour thickness >0.76 mm diagnosed in our Department between 1990 and 1995. The rationale behind this morphological evaluation was based on the hypothesis that a change in the adhesion molecule profile of melanoma cells may manifest visually discrete changes in the way that cells group together in dermal aggregates. We used a dyscohesion score based on the proportion of invasive tumour occupied by dyscohesive neoplastic cells and assessed its clinical significance by correlating it with the incidence of recurrence, regional and distant metastases and survival of the patients. Follow-up was up to 12 years. We found that the degree of melanoma cell dyscohesion was associated with the probability of local recurrence or metastasis. This correlation was particularly significant when dyscohesion involving an area smaller than 25% (dyscohesion score 1) of the dermal component was compared to dyscohesion involving a larger area (scores 2–4). Melanomas in the latter group had significantly increased likelihood of recurrence or metastasis (P

The microtubule-associated protein light chain 3 MAP1LC3 (LC3) and GABARAPL1 and 2 are the major proteins to monitor autophagy. Here, we analyzed the role of both MAP1LC3, GABARAPL1 and 2 in breast cancer cells and primary human breast cancer. HER2 amplified (BT474, SKBR3, MDA-MB-453), ER positive (MCF7, T47D) and triple negative (MDAMB231) breast cancer cell lines were studied in response to hypoxia, in high [25mM] and low glucose [5mM] treatment. Low glucose increased the basal levels for LC3A-II, LC3B-II and GABARAPL2-II in BT474, MCF7 and MDAMB231 cell lines. SKBR3 cell line had the lowest autophagic protein levels for LC3B-II, and Gabarapl2-II and was the most resistance to the autophagy inhibition mediated using siRNAs to all theseIn hypoxia both LC3B (up to 8 fold) and GABARAPL1 (up to 12 fold) mRNA expression were induced in all cell lines (p Co-expression analysis of autophagic genes define distinctive clusters of MAP1LC3, GABARAPL 1 and 2 with the hypoxic signature in breast cancer subtypes using the METABRIC series. The expression levels of the MAP1LC3 and GABARAPL1 and 2 among the breast cancer subtypes and normal tissue showed significant suppession of all of them in cancer compared to normal breast tissue. In conclusion, all the pathways were suppressed at RNA level in cancer compared to normal, baseline levels were not associated with core classification of breast cancer types. These latter data were confirmed in a breast cancer cell line panel, which showed a marked variation in inducibility by environemntal stress and role in survival under low glucose and hypoxia. Thus analysis of induced gene expression in response to stress may be important, or heterogeneity in selection of tumors for anti-autophagy therapy. Citation Format: Christos E. Zois, Syed Haider, Simon Wigfield, Alexandra Giatromanolaki, Efthimios Sivridis, Karl Morten, Ioannis Roxanis, Russell Leek, Francesca Buffa, Michael Koukourakis, Adrian L. Harris. Differential regulation of LC3 A and B, GABARAPL 1 and 2 autophagy genes by micro-environmental stress and role in breast cancer survival. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2903. doi:10.1158/1538-7445.AM2015-2903

In early-onset myasthenia gravis (EOMG), the thymus usually shows medullary lymph node-type infiltrates, rearranged bands of hyperplastic epithelium and focal fenestrations in the intervening laminin borders. This resemblance to autoimmune target organs may reflect autoantigen expression by rare thymic myoid cells, long ago implicated circumstantially as agents provocateurs . In this quantitative study, they were frequently seen at the laminin fenestrations; if so, germinal centers (GC) were significantly commoner nearby, our most EOMG-specific finding (not seen in a distinct MG patient subset). As autoantibodies became detectable, myoid cell involvement apparently progressed. Our unifying hypothesis—that an early autoantibody attack on myoid cells provokes local GC formation—helps to resolve many puzzles.

Background: HER3 is known to locate in the nucleus. Unlike HER4, nuclear HER3 translocation has not been reported to be due to a proteolytic cleavage process by ADAM17 and gamma-secretase. The mechanisms of nuclear HER3 induction and its role in relation to trastuzumab treatment and resistance for HER2-positive breast cancer is unclear. Methods: Using nuclear fractionation and confocal microscopy, nuclear HER3 localisation was investigated in response to trastuzumab with or without ADAM17 inhibitor and gamma-secretase inhibitor in a panel of HER2 expressing cell lines. We also correlated nuclear HER3 expression by immunohistochemistry with treatment response in patients who underwent window trastuzumab study as well as the survival outcome in a cohort of HER2-positive breast cancer patients using Kaplan–Meier survival curves with Log-rank test. Results: HER3 ligand heregulin and trastuzumab was found to induce nuclear HER3 translocation in HER2-positive breast cancer cell lines, including SKBR3. Nuclear HER3 was also enriched in acquired trastuzumab resistant SKBR3 cells (SKBr3-TR). Trastuzumab treatment induced several HER3 fragments and HER3100kD was found to be responsible for nuclear HER3 enrichment by fractionation. This fragment was confirmed to be a specific band of HER3 as shown by HER3 knockdown. Nuclear HER3 was reduced by inhibiting either gamma-secretase or ADAM17 inhibitor. Gamma-secretase or ADAM17 inhibitor reduced HER3100kD in both SKBr3 and SKBr3-TR cells. In HER2-positive breast cancer patients who underwent window trastuzumab study, baseline nuclear HER3 status was not a predictor of response for trastuzumab monotherapy at day 21. However, nuclear HER3 was enriched after trastuzumab treatment in a poor-responder patient. Total HER3 expression level in cytoplasm positively was correlated with poor response to trastuzumab monotherapy in HER2-positive patients (r = 0.67, p = 0.05). There was no statistically significant difference in disease-free survival between positive and negative nuclear HER3 expression but the number of patients was small (n = 87). Further validation to assess nuclear HER3 expression as a prognostic and predictive biomarker in HER2-positive breast cancer patients undergoing trastuzumab treatment will be assessed in randomized tumour samples from FinHER study. Conclusion: Heregulin and trastuzumab treatment seems to induce nuclear HER3 translocation in some of the HER2 positive breast cancer cells. This may be due to proteolytic cleavage of HER3 as it is reduced by ADAM17 or gamma-secretase inhibitor. Enriched nuclear localisation of HER3 seems to be one possible mechanism of acquired resistance to trastuzumab in HER2-positive breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-05-08.

The early-onset myasthenia gravis (EOMG) thymus shows characteristic medullary epithelial bands (MEB), greatly expanded perivascular infiltrates and fenestrations of the intervening basement membranes. We now compare epithelial expression of epidermal growth factor receptor (EGFR) and many integrins in EOMG and control samples. The main differences are striking/consistent thickening (in MEB) of what is normally a monolayer of perivascular epithelium, with focal protrusion into the infiltrates. This evidently hyperplastic epithelial subpopulation also strongly expresses EGFR and certain integrins. We suggest that its enhanced interactions with the locally increased extracellular matrix protein deposits may play an important role in autosensitization.

The muscle weakness in myasthenia gravis (MG) is clearly mediated by autoantibodies to the acetylcholine receptor (AChR). Their production depends on helper T cells, the induction of which must be a crucial step in autosensitization [1]. MG patients with onset of the disease before 40 years of age (early onset MG-EOMG) typically show a characteristic thymic hyperplasia (reviewed in [1]). This is largely due to invasion of the extraparenchymal perivascular spaces by lymphoid infiltrates with germinal centers (GC) and T cell areas [2–4]. In parallel, the true thymic parenchyma also shows alterations, with rearrangement of the medullary epithelium into bands (medullary epithelial bands-MEB) [3, 4]. In normal thymus, the two compartments are separated by a laminin+ basement membrane. By contrast, in the EOMG thymus, this barrier is frequently fenestrated [3, 4], and thymic epithelial cells (TEC) are occasionally seen protruding toward the lymphoid infiltrates. Such TEC are intermingled with abundant deposits of extracellular matrix (ECM) proteins (e.g. laminin) that are continuous with an analogous network in T cell areas. Furthermore, they express high levels of ECM protein receptors (integrins) and epidermal growth factor receptor (EGFR) [5], which could synergize in leading to TEC proliferation and migration. As a result, in the EOMG thymus, a considerable proportion of TEC is aberrantly exposed to the lymphoid infiltrates. Interestingly, it has been previously noticed that GC are frequently localized at sites of basement membrane fenestrations [3, 4]. There is also recent evidence that TEC express AChR subunits [6], though not whole AChR.

Myasthenia gravis (MG) is caused by helper T cell-dependent autoantibodies against the muscle acetylcholine receptor (AChR). Thymic epithelial tumors (thymomas) occur in 10% of MG patients, but their autoimmunizing potential is unclear. They express mRNAs encoding AChR alpha and epsilon subunits, and might aberrantly select or sensitize developing thymocytes or recirculating peripheral T cells against AChR epitopes. Alternatively, there could be defective self-tolerance induction in the abundant maturing thymocytes that they usually generate. For the first time, we have isolated and characterized AChR-specific T cell clones from two MG thymomas. They recognize extracellular epitopes (alpha75-90 and alpha149-158) which are processed very efficiently from muscle AChR. Both clones express CD4 and CD8alpha, and have a Th-0 cytokine profile, producing IL-4 as well as IFN-gamma. They are restricted to HLA-DP14 and DR52a; expression of these minority isotypes was strong on professional antigen-presenting cells in the donors' tumors, although it is generally weak in the periphery. The two clones' T cell receptor beta chains are different, but their alpha chain sequences are very similar. These resemblances, and the striking contrasts with T cells previously cloned from non-thymoma patients, show that thymomas generate and actively induce specific T cells rather than merely failing to tolerize them against self antigens.

It has long been recognised that autoimmunity may be associated with particular tumours, and the regular co-occurrence of myasthenia gravis (MG) with thymoma is well established. In this brief review we will summarise some of the evidence for thymoma-associated antibodies in myasthenia, and in other neurological disorders, and discuss the possible relationship between the tumour, these antibodies and the initiation of the disease.

Background. Trastuzumab is used in HER2 positive breast cancer patients and has been shown to increase survival in these patients. However, drug resistance imposes a crucial limitation to the successful treatment of HER2 positive breast cancer. Multiple mechanisms have been proposed but the role of HER4 in relation to Trastuzumab treatment and resistance remains unclear. This study aims to investigate the role of HER4 in relation to Trastuzumab treatment and resistance in HER2 positive breast cancer. Methods. The effects of Trastuzumab on the protein and mRNA levels of HER4 were studied in both naïve and Trastuzumab resistant cells using western blot and RT-PCR. HER4 localisation after Trastuzumab treatment was determined by confocal microscopy and nuclear fractionation. A gamma-secretase inhibitor (GSI) and a pan-HER kinase inhibitor, Neratinib, were used to inhibit HER4 cleavage during Trastuzumab treatment. The role of HER4 was further validated using siRNA experiments. HER4 level and its localisation were also assessed by IHC in BT474 xenograft samples treated with Trastuzumab or control. To understand the prognostic role of HER4, TMAs of a cohort of HER2 positive breast cancer patients were stained for HER4 expression using IHC and the levels were correlated with patients’ outcome. Results. The basal level of HER4 in HER2 positive SKBR3 and BT474 is comparable to HER2 negative MCF7 cells. In SKBR3 and BT474, Trastuzumab treatment upregulated HER4 mRNA and protein expression in a dose responsive manner. Moreover, HER480kDa was also increased and an enhanced nuclear localisation was observed, indicating that Trastuzumab promotes HER4 cleavage. Resistant cells appear to have more nuclear HER4 and Trastuzumab withdrawal causes a decrease in nuclear intensity. HER4 downregulation via siRNA reduces cell viability in Trastuzumab sensitive and resistant cells. In SKBR3, GSI decreased HER4 nuclear staining and decreased Trastuzumab induced HER4 nuclear translocation. In addition, GSI also enhanced response to Trastuzumab in decreasing cell viability. Neratinib also blocks HER480kDa formation induced by Trastuzumab, and combination treatment of Trastuzumab and Neratinib had an additive inhibition effect on cell viability. There was a significant upregulation of nuclear HER4 in a Trastuzumab treated BT474 xenograft model compared to the control. Nuclear HER4 was also prognostic in a cohort of HER2 positive breast cancer patients. Conclusion. This study shows that Trastuzumab upregulates HER4 in vitro and in vivo. Nuclear HER4 seems to mediate Trastuzumab resistance in HER2 positive breast cancer cells. HER4 knockdown as well as drug treatments that decrease nuclear HER4 may enhance sensitivity to Trastuzumab. We believe that the prognostic and predictive values of nuclear HER4 in relation to Trastuzumab resistance need to be further validated in HER2 positive breast cancer patients. Citation Format: Siti Norasikin Mohd Nafi, Merel Gijsen, Ioannis Roxanis, Gabriela Kramer-Marek, Jacek Capala, Anthony Kong. HER4 cleavage and nuclear localization mediate Trastuzumab resistance in HER2-positive breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 531. doi:10.1158/1538-7445.AM2013-531

Background The term Metacognition refers to the psychological processes that are involved in the way a person controls, modifies, and appraises his own thoughts [1]. Maladaptive metacognitions have been related to the development and maintenance of psychological disorder [2,3]. The Metacognitions Questionnaire-30 (MCQ-30) is a multidimensional instrument for assessing metacognitions, composed of five factors: cognitive confidence, positive beliefs about worry, cognitive self consciousness, negative beliefs about worry and need to control thoughts [4]. Psychometric properties of the MCQ-30 have been well documented [4] suggesting that it is a valid instrument that has already been used in clinical research with several psychiatric disorders.