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Tobias Röck,1 Hans-Christian Bösmüller,2 Karl Ulrich Bartz-Schmidt,1 Daniel Röck1 1Centre for Ophthalmology, University of Tübingen, Tübingen, Germany; 2Institute of Pathology and Neuropathology, University of Tübingen, Tübingen, Germany Purpose: Nevi of the conjunctiva are usually benign pigmented tumorous lesions located in the bulbar conjunctiva. In most conjunctival nevus cases, the patient wants the lesion to be removed for cosmetic reasons, but excisional biopsies are best for lesions suspicious for malignancy. This case report illustrates the intraoperative surgical management, histological findings, and the course of healing in a conjunctival nevus patient. Case report: A 26-year-old man was referred to our eye hospital with a large bulbar conjunctival nevus of the right eye. Upon examination, there was a large pigmented lesion with numerous small cysts present on the superior bulbar conjunctiva. The conjunctival tumor was resected, and an amniotic membrane transplantation was performed for the bulbar conjunctival reconstruction. The histopathological diagnosis suggested a conjunctival nevus. After the resection, a reduction in the inflammation and healing of the conjunctival lesion could be seen. The epithelialization of the bulbar conjunctiva over the amniotic membrane was complete 4 weeks after the resection. At the 6-month follow-up, there was no sign of recurrence or any postoperative complications. Conclusion: A surgical excision combined with reconstruction via amniotic membrane transplantation is effective and economical for the treatment of large conjunctival lesions. Keywords: conjunctival nevus, reconstructive surgical procedures, amniotic membrane transplantation, ocular surface reconstruction

BACKGROUND Conjunctival squamous cell carcinoma is the most common non-pigmented malignancy of the ocular surface. This report illustrates the clinical management of squamous cell carcinoma of the conjunctiva. CASE REPORT A 33-year-old female was referred to our eye hospital with a tumorous lesion on the nasal bulbar conjunctiva of the right eye. A topical therapy with antibiotic and corticosteroid eye drops did not change the lesion. The conjunctival tumor was widely resected. The histopathological diagnosis suggested a squamous cell carcinoma. After resection, a treatment with topical mitomycin C (MMC) 0.02% eye drops were started 4 times daily for 14 days. Two cycles of treatment were done with a 2-week interval during which only artificial tears eye drops were administered. At the 12-month follow-up, there was no sign of recurrence. CONCLUSIONS This case illustrates the effective and successful clinical management of squamous cell carcinoma of the conjunctiva with excision and postoperative treatment with MMC 0.02% eye drops.

Breast cancer is the second leading cause of cancer death among women worldwide and besides life style, age and genetic risk factors, exposure to ionizing radiation is known to increase the risk for breast cancer. Further, DNA copy number alterations (CNAs), which can result from radiation-induced double-strand breaks, are frequently occurring in breast cancer cells. We set out to identify a signature of CNAs discriminating breast cancers from radiation-exposed and non-exposed female patients. We analyzed resected breast cancer tissues from 68 exposed female Chernobyl clean-up workers and evacuees and 68 matched non-exposed control patients for CNAs by array comparative genomic hybridization analysis (aCGH). Using a stepwise forward-backward selection approach a non-complex CNA signature, that is, less than ten features, was identified in the training data set, which could be subsequently validated in the validation data set (p value < 0.05). The signature consisted of nine copy number regions located on chromosomal bands 7q11.22-11.23, 7q21.3, 16q24.3, 17q21.31, 20p11.23-11.21, 1p21.1, 2q35, 2q35, 6p22.2. The signature was independent of any clinical characteristics of the patients. In all, we identified a CNA signature that has the potential to allow identification of radiation-associated breast cancer at the individual level., (© 2018 UICC.)

Ionizing radiation is a well-recognized risk factor for the development of breast cancer. However, it is unknown whether radiation-specific molecular oncogenic mechanisms exist. We investigated post-Chernobyl breast cancers from radiation-exposed female clean-up workers and nonexposed controls for molecular changes. Radiation-associated alterations identified in the discovery cohort (n = 38) were subsequently validated in a second cohort (n = 39). Increased expression of hsa-miR-26b-5p was associated with radiation exposure in both of the cohorts. Moreover, downregulation of the TRPS1 protein, which is a transcriptional target of hsa-miR-26b-5p, was associated with radiation exposure. As TRPS1 overexpression is common in sporadic breast cancer, its observed downregulation in radiation-associated breast cancer warrants clarification of the specific functional role of TRPS1 in the radiation context. For this purpose, the impact of TRPS1 on the transcriptome was characterized in two radiation-transformed breast cell culture models after siRNA-knockdown. Deregulated genes upon TRPS1 knockdown were associated with DNA-repair, cell cycle, mitosis, cell migration, angiogenesis and EMT pathways. Furthermore, we identified the interaction partners of TRPS1 from the transcriptomic correlation networks derived from gene expression data on radiation-transformed breast cell culture models and sporadic breast cancer tissues provided by the TCGA database. The genes correlating with TRPS1 in the radiation-transformed breast cell lines were primarily linked to DNA damage response and chromosome segregation, while the transcriptional interaction partners in the sporadic breast cancers were mostly associated with apoptosis. Thus, upregulation of hsa-miR-26b-5p and downregulation of TRPS1 in radiation-associated breast cancer tissue samples suggests these molecules representing radiation markers in breast cancer., (© 2017 UICC.)

Immunotherapies, particularly checkpoint inhibitors, have set off a revolution in cancer therapy by releasing the power of the immune system. However, only little is known about the antigens that are essentially presented on cancer cells, capable of exposing them to immune cells. Large-scale HLA ligandome analysis has enabled us to exhaustively characterize the immunopeptidomic landscape of epithelial ovarian cancers (EOCs). Additional comparative profiling with the immunopeptidome of a variety of benign sources has unveiled a multitude of ovarian cancer antigens (MUC16, MSLN, LGALS1, IDO1, KLK10) to be presented by HLA class I and class II molecules exclusively on ovarian cancer cells. Most strikingly, ligands derived from mucin 16 and mesothelin, a molecular axis of prognostic importance in EOC, are prominent in a majority of patients. Differential gene-expression analysis has allowed us to confirm the relevance of these targets for EOC and further provided important insights into the relationship between gene transcript levels and HLA ligand presentation., Competing Interests: Conflict of interest statement: H.-G.R. is a shareholder of Immatics Biotechnologies, Tübingen, and CureVac GmbH, Tübingen. H.S. and D.J.K. are employees of Immatics Biotechnologies GmbH. The authors declare that Immatics did not provide neither financial nor scientific support in any direct relation to this manuscript or the underlying studies, and was not involved in data collection, analysis, or decision to publish., (Copyright © 2017 the Author(s). Published by PNAS.)

Objective: Neural cell adhesion molecule (CD56) has been proposed as a potential marker for neuroendocrine differentiation in carcinomas, together with synaptophysin and chromogranin A. However, CD56 immunoreactivity by itself can be found in a broad variety of tumors, including ovarian neoplasms. CD56 has recently been suggested as a potential target for antibody-based therapy. However, for ovarian carcinoma, there is only limited data available regarding the pattern of CD56 immunoreactivity, coexpression of neuroendocrine markers, and correlation with histological types and clinical parameters., Methods: In our study, we therefore evaluated CD56 staining by immunohistochemistry on a tissue micrroarray with 206 ovarian carcinomas, including 151 high-grade serous, 7 low-grade serous, 32 endometrioid, 11 clear cell, 5 mucinous, as well as 33 atypically proliferating serous tumors/serous borderline tumors., Results: At least focal CD56 immunoreactivity was observed in 65% of carcinomas of all histological types. Moderate staining with at least 10% positive cells was found in 44 (28%) high-grade serous carcinomas (HGSOCs), 2 (29%) low-grade serous and 3(9%) endometrioid carcinomas. Strong immunoreactivity was limited to 10 (7%) HGSOCs. There was no correlation with the expression of chromogranin or synaptophysin. Serous borderline tumors showed only weak and focal staining in 11 (33%). Expression of CD56 overall was significantly associated with high-grade and advanced stage. In the subgroup of HGSOCs, CD56 expression was associated with reduced overall survival (median 30 vs. 47 months, P = 0.039, log rank, univariate analysis)., Conclusions: CD56 (neural cell adhesion molecule) is frequently expressed in ovarian carcinomas and is significantly associated with HGSOC and advanced tumor stage. Due to its lack of correlation with neuroendocrine differentiation, CD56 expression is of limited diagnostic value, but may rather serve as a marker for tumor progression or as a potential therapeutic target.

Objective: Increased numbers of tumor-infiltrating lymphocytes (TILs) in high-grade serous ovarian cancer (HGSC) are associated with improved clinical outcome. Intraepithelial localization of TILs might be regulated by specific homing receptors, such as CD103, which is widely expressed by intraepithelial lymphocytes. Given the emerging role of CD103 TILs, we aimed to assess their contribution to the prognostic value of immunoscoring in HGSC., Methods: The density of intratumoral CD3 and CD103 lymphocytes was examined by immunohistochemistry on a tissue microarray of a series of 135 patients with advanced HGSC and correlated with CD4, CD8, CD56, FoxP3, and TCRγ T-cell counts, as well as E-cadherin staining and conventional prognostic parameters and clinical outcome., Results: Both the presence of CD103 cells, as well as high numbers of intraepithelial CD3 lymphocytes (CD3E), showed a significant correlation with overall survival, in the complete series, as well as in patients with optimal debulking and/or platinum sensitivity. Combining CD3 and CD103 counts improved prognostication and identified 3 major subgroups with respect to overall survival. The most pronounced effect was demonstrated for patients with optimally resected and platinum-sensitive tumors. Patients with CD3/CD103 tumors showed a 5-year survival rate at 90%, CD3/CD103 at 63%, and CD3/CD103 at 0% (P < 0.001)., Conclusions: These results suggest that combined assessment of CD103 and CD3 counts improves the prognostic value of TIL counts in HGSC and might identify patients with early relapse or long-term survival based on the type and extent of the immune response.

The recent approval of clincially effective immune checkpoint inhibitors illustrates the potential of cancer immunotherapy. A challenging task remains the identification of specific targets guiding immunotherapy. Facilitated by technical advances, the direct identification of physiologically relevant targets is enabled by analyzing the HLA ligandome of cancer cells. Since recent publications demonstrate the immunogenicity of ovarian cancer (OvCa), immunotherapies, including peptide-based cancer vaccines, represent a promising treatment approach. To identify vaccine peptides, we employed a combined strategy of HLA ligandomics in high-grade serous OvCa samples and immunogenicity analysis. Only few proteins were naturally presented as HLA ligands on all samples analyzed, including histone deacetylase (HDAC) 1 and 2. In vitro priming of CD8(+) T cells demonstrated that two HDAC1/2-derived HLA ligands can induce T-cell responses, capable of killing HLA-matched tumor cells. High HDAC1 expression shown by immunohistochemistry in 136 high-grade serous OvCa patients associated with significantly reduced overall survival (OS), whereas patients with high numbers of CD3(+) tumor-infiltrating lymphocytes (TILs) in the tumor epithelium and CD8(+) TILs in the tumor stroma showed improved OS. However, correlating HDAC1 expression with TILs, high levels of TILs abrogated the impact of HDAC1 on OS. This study strengthens the role of HDAC1/2 as an important tumor antigen in OvCa, demonstrating its impact on OS in a large cohort of OvCa patients. We further identified two immunogenic HDAC1-derived peptides, which frequently induce multi-functional T-cell responses in many donors, suitable for future multi-peptide vaccine trials in OvCa patients.

Recently, the occurrence of cyclin D1-positive B cells with mantle cell lymphoma phenotype in the inner mantle zones of morphologically inconspicuous lymph nodes has been described and termed mantle cell lymphoma 'in situ'. Prevalence and clinical significance of this lesion and related minimal mantle cell lymphoma infiltrates in reactive lymphoid tissues of healthy individuals, and of mantle cell lymphoma patients are unknown. All 1292 reactive lymph nodes from unselected consecutive surgical specimens of 131 patients without a history of lymphoma obtained over a 3-month period were stained for cyclin D1. In addition, all morphologically reactive lymph nodes and benign-appearing extranodal lymphoid infiltrates of patients diagnosed with mantle cell lymphoma in the years 2000-2011 were studied. Samples predating the lymphoma diagnosis for at least 2 months were available from 37/423 (9%) patients. A mantle cell lymphoma 'in situ' was not identified in any of the two groups. However, in four patients with subsequent mantle cell lymphoma diagnosis, an early manifestation of mantle cell lymphoma was detected retrospectively, antedating the lymphoma diagnosis for 2-86 months. In six mantle cell lymphoma patients, only small groups of cyclin D1-positive cells in morphologically reactive extranodal infiltrates were detected >2 months before the diagnosis of mantle cell lymphoma (range 3-59 months). Mantle cell lymphoma 'in situ' is an extremely rare phenomenon in morphologically reactive lymph nodes, in line with the low prevalence of t(11;14)-positive cells described in the peripheral blood of a healthy population. In mantle cell lymphoma patients, however, immunohistochemically detectable infiltrates of mantle cell lymphoma cells antedating the lymphoma diagnosis were found in a significant proportion of cases (10/37=27%). These consisted either of early mantle cell lymphoma with mantle zone growth pattern, or small extranodal accumulations of cyclin D1+ cells, whereas typical mantle cell lymphoma 'in situ' was not detected.

The aim of this study was to investigate the molecular and protein expression pattern of markers of stemness phenotype and its clinicopathological significance in human biliary tract cancer (BTC). Human BTC cell lines (CCLP-1, Egi-1, MzChA-1, MzChA-2, SkChA-1, TFK-1 and GBC) were analyzed in vitro and in xenotransplanted animals for expression of markers of stemness and compared to tissue microarrays (TMA) of 34 cases of human BTC with complete pathomorphological and clinical data (survival). Molecular analyses on the mRNA and protein level included makers of stemness and progenitor (Bmi-1, Sox-2, Nestin, CD133, CD44 and Nanog), proliferation and differentiation (cell cycle proteins, intermediate filaments). The investigated BTC samples showed a low to moderate and partially significantly different expression pattern of the stem cell markers in vitro, in vivo and in TMA. Hierarchical cluster analysis identified subgroups with homogenous expression of stem cell markers significantly differing with respect to cytokeratin expression in xenografts and Ki67 proliferation marker in human TMA, respectively - thus indicating possible heterogeneous carcinogenesis pathways in BTC. Additionally, these stem cell markers could be linked to morphology and molecular markers of proliferation and differentiation on the mRNA and protein level. Finally, survival analysis identified the combination of CD133 and CD44 as an independent prognostic factor yet their value as prognostic factors need testing in prospective study design.

Suppressors of cytokine signalling (SOCS) and protein tyrosine phosphatase (PTPN) proteins are negative regulators of Janus Kinase 2 (JAK2). They are thought to be involved in the molecular pathogenesis of essential thrombocythaemia (ET) particularly in patients with unmutated JAK2. In this study we compared DNA methylation of SOCS1, SOCS3 and PTPN6 in peripheral blood cells between 39 ET patients (24 JAK2 V617F mutated) and 22 healthy controls by methylation specific PCR (MSP) and analysed the clinical outcome of patients with respect to DNA methylation. In SOCS1, ET patients showed significantly less methylation (P<0.05) than healthy controls, and in SOCS3 and PTPN6 such a tendency was shown. However, there were no significant differences in the methylation frequencies between JAK2 wildtype and mutated ET patients. In addition, no correlation was detected between methylation of SOCS and PTPN and any clinical outcome parameters. Taken together, regarding the genomic regions investigated our data indicate a minor role of methylation of JAK2 negative regulators for the clinical course of ET., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Diagnosis of infections caused by mycobacteria, especially nontuberculous mycobacteria still represents a difficult task both in microbiology and pathology. The aim of this study was to determine the frequency of mycobacterial DNA detectable by PCR in formalin-fixed paraffin-embedded tissues showing suspicious granulomatous lesions. A total of 190 archival specimens were analyzed, using a nested PCR protocol, which amplifies a fragment of the mycobacterial 65-kDa heat-shock protein gene. Restriction fragment-length polymorphisms and sequencing were utilized to further analyze the obtained PCR products. Corresponding microbiological culture results were available for 41 cases. We detected mycobacterial DNA in 119 cases (63%), of which 71 (60%) were positive for Mycobacterium tuberculosis complex DNA and 41 (34%) for DNA of nontuberculous mycobacteria. Seven cases (6%) could not be subtyped for technical reasons. The largest group of nontuberculous mycobacteria comprised 29 cases (25% of the 119 positive cases), which were assigned to Mycobacterium fortuitum complex. Mycobacterium avium-intracellulare complex was detected in eight (7%) cases, Mycobacterium gordonae in three (2.5%) and Mycobacterium rhodesiae in a single case (0.8%). All cases of Mycobacterium tuberculosis were unequivocally identified by restriction fragment-length polymorphism analysis. In contrast, sequencing provided a gain of information over restriction fragment-length polymorphism analysis in 37% of the nontuberculous mycobacteria cases (15 of 41). Alignment studies on DNA of nontuberculous mycobacteria showed frequent sequence variations, supporting the existence of sequevars. Comparison of molecular data to available results of microbiological culture assays showed a good concordance of 83%. In conclusion, amplification and sequencing of the mycobacterial 65-kDa heat-shock protein gene is an excellent tool for species identification of mycobacteria, especially nontuberculous mycobacteria, in formalin-fixed paraffin-embedded tissues.

This report describes the clinico-pathological features of myofibroblastic tumors of the breast in six patients. Four women and one man presented with a benign myofibroblastoma. The sixth patient was a woman with myofibrosarcoma. All myofibroblastomas were composed of a fascicular arrangement of spindle cells embedded in dense bundles of collagen. Tumors differed with respect to their proportion of neoplastic cells and collagenous stroma as well as cellular pleomorphism. Based on this variation, the tumors could be subclassified as classic, collagenized, epithelioid and cellular myofibroblastoma. Immunohistological staining confirmed myofibroblastic differentiation by strong expression of either desmin or smooth muscle actin with coexpression of vimentin. In addition, numerous cells reacted with antibodies to CD68. Proliferative activity was rather low in the myofibroblastoma with an average of 0-2 mitotic figures per 10 HPF. DNA cytometric analysis was performed in two cases and showed diploid stem lines with minor S-phase fractions (1% and 3%). In the myofibrosarcoma, cells contained pleomorphic nuclei with some giant cells and numerous mitotic figures (6-7/10 HPF) and had infiltrating margins that were apparent even grossly. Immunohistochemically, tumor cells strongly expressed vimentin, smooth muscle actin and fibronectin. Ultrastructurally, neoplastic cells met the criteria of myofibroblasts, i.e. contained abundant intermediate filaments and myofilament bundles with focal densities as well as fibronexus junctions. DNA cytometric analysis exhibited again a diploid stemline but marked proliferative activity was present as indicated by an S-phase fraction of 20%. In conclusion, in benign myofibroblastoma there may be some cellular pleomorphism but mitotic activity is always low. The malignant counterpart, myofibrosarcoma, is characterized by marked cellular pleomorphism, infiltrating margins and high mitotic rate.