[Uveal Melanoma Cell Under Oxidative Stress - Influence of VEGF and VEGF-Inhibitors]. / Uveale Melanomzellen unter oxidativem Stress ­ Einfluss von VEGF und VEGF-Inhibitoren.

BACKGROUND: The role of oxidative stress in cancer is complex. While the pathological alterations induced by oxidative stress may be involved in the induction of tumours, in the late stages of tumour development, it can facilitate the loss of tumour cells and might even prevent metastasis. Tumour cells show metabolic alterations, often inducing an increased production of reactive oxygen species, which makes these cells particularly vulnerable to additional oxidative stress. This is an important mode of action in the use of many chemotherapeutics and in the application of ionizing radiation. Uveal melanoma is the most frequent primary tumour in the adult eye. For metastasis of this tumour, which affects about 50 % of the patients, no appropriate treatment is currently available. However, the primary tumour can efficiently be treated with ionizing radiation. A frequent side effect of this treatment is radiation retinopathy, which is treated with vascular endothelial growth factor (VEGF) antagonists. A therapy of the primary tumour with VEGF antagonists is under discussion. So far, little data is available on this subject, however, a paradoxical worsening of the situation has been found in a mouse model of uveal melanoma treated with bevacizumab. METHODS: We have investigated the effect of VEGF and of the VEGF-antagonist bevacizumab on the survival of five different melanoma cell lines under oxidative stress treatment with hydrogen peroxide. In addition, we investigated the expression of relevant proteins and the effect of bevacizumab on the proliferation of the cells as well as its effect on the angiogenic behaviour of endothelial cells, co-cultured with uveal melanoma cells. RESULTS: Our study showed that not only VEGF but also, paradoxically, the VEGF-antagonist bevacizumab is able to protect uveal melanoma cells from oxidative stress-induced cell death. Bevacizumab did not influence the proliferation of the cells and showed only limited effectiveness to reduce angiogenic structures. CONCLUSION: Considering that oxidative stress is the mode of action for ionizing radiation to induce cell death, a protective effect of bevacizumab on uveal melanoma cells against oxidative stress is worrisome and argues against the use of VEGF in uveal melanoma.

Electrochemotherapy with bleomycin and cisplatin enhances cytotoxicity in primary and metastatic uveal melanoma cell lines in vitro.

Electrochemotherapy (ECT) enhances responsiveness to cytotoxic drugs in numerous cell lines in vitro. Clinically ECT is widely applied for skin tumor ablation and has shown efficacy in treating non-resectable colorectal liver metastases. There is limited experience of ECT for ocular tumor therapy. We investigated the cytotoxic effect of bleomycin and cisplatin in combination with electroporation on chemoresistant human uveal melanoma (UM) cell lines in vitro. Four UM cell lines (Mel 270, 92-1, OMM-1, OMM-2.5) were treated with electroporation (pulse amplitude 300-1000 V/cm, 8-80 pulses, 100 µs, 5 Hz) and increasing concentrations of bleomycin and cisplatin (0-7.5 µg/ml). Cell survival was analyzed by MTT viability assay after 36 hours. UM cell lines were resistant to both bleomycin and cisplatin. In combination with electro- poration, the effects of bleomycin and cisplatin were increased 8-70 fold and 3-15 fold, respectively, in all UM cell lines. At the lowest concentration of bleomycin tested (1 µg/ml), viability was maximally reduced in all UM cell lines by ≥69% with electroporation conditions of 750 V/cm and 20 pulses. All UM cell lines were more resistant to cisplatin; however, electro- poration of 1000 V/cm and 8 pulses resulted in similar reductions in cell viability of 92-1, Mel270 with 2.5 µg/ml cisplatin, OMM2-5 cells with 5 µg/ml cisplatin and OMM1 cells with 1 µg/ml cisplatin. In vitro ECT with bleomycin or cisplatin is more effective than the highest concentration of the antineoplastic drug or electroporation alone, opening new perspectives in primary and metastatic UM treatment.

[Malignant lymphomas of the eye]. / Maligne Lymphome des Auges.

The eye and the ocular adnexae are rare sites for malignant non-Hodgkin lymphoma (NHL). Based on their anatomical location, intraocular lymphomas must be discerned from NHL of adnexal structures including conjunctiva, lacrimal gland, and orbit. Whereas the latter group mostly consists of indolent extranodal marginal zone B­cell lymphomas of mucosa-associated lymphoid tissue (MALT) type or secondary manifestations of systemic NHL, most primary intraocular lymphomas are classified as diffuse large B­cell lymphomas (DLBCL) and are considered a variant of primary DLBCL of the central nervous system. The most common form is primary vitreoretinal lymphoma (PVRL), which presents with nonspecific symptoms and is difficult to discern from uveitis. Diagnosis of PVRL is usually made by cytological, immunocytochemical, and molecular analysis of vitreous aspirates. Degenerative changes, limited material, and the occurrence of pseudoclonality in the molecular analysis of B­cell clonality can hamper diagnostic assessment. Novel techniques such as detection of MYD88 mutations common in PVRL can increase diagnostic sensitivity. Close cooperation with clinical colleagues and rapid specimen processing are fundamental for successful diagnosis.

[Ocular melanomas : An update]. / Okuläre Melanome : Ein "Update".

Melanoma is the most common type of primary cancer to affect the adult eye. Approximately 95% of ocular melanomas are intraocular and arise from the uvea (i. e. iris, ciliary body, and choroid), while the remaining 5% are located in the conjunctiva. Although both uveal and conjunctival melanomas are thought to derive from malignantly transformed melanocytes, uveal melanoma is clinically and biologically distinct from conjunctival melanoma, and indeed from its more common cutaneous counterpart. Intense efforts have been recently made to understand the molecular biology involved in the development of ocular melanomas, and in their progression. Molecular advances, particularly for uveal melanoma, have enhanced prognostication and the identification of rational therapeutic targets for disseminated disease. In this review, recent advances in the molecular characterisation of both uveal and conjunctival melanomas are discussed, and how these may be used to develop personalised therapeutic strategies.

The epidemiology, clinical characteristics, histopathology and management of juvenile- and adult-onset corneoscleral limbus xanthogranuloma.

PURPOSE: Adult-onset xanthogranuloma (AOX) of the corneoscleral limbus is a rare inflammatory condition of unknown aetiology. Similar to limbal juvenile xanthogranuloma (JXG), it presents as a growing mass at the corneoscleral junction. Limbal AOX and JXG can lead to sight-threatening complications if not managed in a timely manner. This systematic review summarises the main clinical and histopathological features of limbal AOX/JXG and discusses the management of this uncommon disease. METHODS: We performed a literature search in the MEDLINE database for all historical entries, using the search terms "limbus", "limbal" and "xanthogranuloma", and retrieved all articles reporting on limbal xanthogranuloma. After refining the search to articles relevant to limbal AOX, we were able to identify ten adult cases of limbal AOX and compare those with all reported cases of limbal JXG. RESULTS: Clinically, AOX usually presents as an isolated smooth, yellowish, dome-shaped nodule at the corneoscleral junction, similar to an ocular presentation of JXG, with which it also shares similar histopathological features. CONCLUSION: Limbal JXG and AOX may represent the same disease entity. Diagnosis relies on the clinical presentation, pathology and immunohistochemical profile. Spontaneous regression is unlikely, and thus prompt surgical intervention should be considered to prevent sight-threatening complications. Xanthogranuloma should be included in the differential diagnosis of corneoscleral limbal masses in patients of all age groups.

Conjunctival squamous cell neoplasia: the Liverpool Ocular Oncology Centre experience.

PURPOSE: To report the outcome of patients with conjunctival squamous cell neoplasia (CSCN)--including conjunctival squamous cell carcinoma (SCC), conjunctival squamous intraepithelial neoplasia (C-SIN) and carcinoma in situ (CIS)-treated at the Liverpool Ocular Oncology Centre (LOOC). METHODS: Patients treated between January 1993 and September 2011 were identified and categorised as having 'primary' or 'salvage' treatment, according to whether they had undergone a surgical procedure before referral to our centre. Invasive SCC was treated by excision with adjunctive ruthenium plaque radiotherapy. C-SIN or CIS was treated with topical 5-fluorouracil (5-FU), and in a few cases, cryotherapy. RESULTS: Primary treatment was administered to 20 patients (16 males, four females). Mean age was 62 years (range, 33-85). Histological examination revealed C-SIN/CIS in ten patients and invasive SCC in nine. Median follow-up was 69 months (range, 34-168). Three patients required further topical chemotherapy for persistent/recurrent C-SIN. Salvage therapy was administered to 21 patients (15 males, six females). Mean age was 63 years (range, 26-82). Histology showed C-SIN/CIS in 11 patients and invasive SCC in ten. Median follow-up was 54.5 months (range, 36-120). At the close of this audit, there was no recurrence of invasive or metastatic disease in either the primary or salvage groups. CONCLUSIONS: Our established protocol for treatment of CSCN has proven successful in local tumour control, and avoids ocular complications. We advocate adjunctive radiotherapy in patients with invasive SCC and chemotherapy in C-SIN/CIS. For improved patient outcome, prompt referral to a specialist centre is encouraged.

Lack of BAP1 protein expression in uveal melanoma is associated with increased metastatic risk and has utility in routine prognostic testing.

BACKGROUND: The absence of BRCA1-associated protein 1 (BAP1) expression in uveal melanoma (UM) is associated with metastatic progression and reduced survival. In this study, we examine nuclear BAP1 (nBAP1) protein expression in primary UMs (PUMs) that show both 'typical' and 'atypical' clinical courses according to their chromosome 3 status, and secondary hepatic metastatic UM (MUM), correlating the results with histological, clinical and survival data. METHODS: Nuclear BAP1 expression was immunohistochemically assessed in tissue microarrays (TMAs) of: (a) 68 PUM patients, who had been treated surgically; and (b) 13 MUM patients, with 5 cases being paired with primary tumour tissue. All cases were fully annotated. The percentage of tumour cell nuclei staining positively for BAP1 was scored by independent observers. RESULTS: Nuclear BAP1 protein expression was absent in 35 out of 68 (51%) PUM patients, correlating strongly with poor prognostic clinicopathological and genetic parameters and reduced survival (Log rank, P<0.001). Lack of nBAP1 expression importantly identified a subset of 'atypical' PUM patients with disomy of chromosome 3 but with unexpected metastatic relapse. Nuclear BAP1 expression was absent in 10 out of 13 (77%) MUM and expression was concordant in all paired PUM and MUM patients. CONCLUSIONS: Absent nBAP1 protein expression is an independent survival predictor for UM patients, easily examined using immunohistochemistry.

Targeting SRPK1 to control VEGF-mediated tumour angiogenesis in metastatic melanoma.

BACKGROUND: Current therapies for metastatic melanoma are targeted either at cancer mutations driving growth (e.g., vemurafenib) or immune-based therapies (e.g., ipilimumab). Tumour progression also requires angiogenesis, which is regulated by VEGF-A, itself alternatively spliced to form two families of isoforms, pro- and anti-angiogenic. Metastatic melanoma is associated with a splicing switch to pro-angiogenic VEGF-A, previously shown to be regulated by SRSF1 phosphorylation by SRPK1. Here, we show a novel approach to preventing angiogenesis-targeting splicing factor kinases that are highly expressed in melanomas. METHODS: We used RT-PCR, western blotting and immunohistochemistry to investigate SRPK1, SRSF1 and VEGF expression in tumour cells, and in vivo xenograft assays to investigate SRPK1 knockdown and inhibition in vivo. RESULTS: In both uveal and cutaneous melanoma cell lines, SRPK1 was highly expressed, and inhibition of SRPK1 by knockdown or with pharmacological inhibitors reduced pro-angiogenic VEGF expression maintaining the production of anti-angiogenic VEGF isoforms. Both pharmacological SRPK1 inhibitors and SRPK1 knockdown reduced growth of human melanomas in vivo, but neither affected cell proliferation in vitro. CONCLUSIONS: These results suggest that selective blocking of pro-angiogenic isoforms by inhibiting splice-site selection with SRPK1 inhibitors reduces melanoma growth. SRPK1 inhibitors may be used as therapeutic agents.

The Liverpool uveal melanoma liver metastases pathway: outcome following liver resection.

AIM: To determine the outcome of patients that underwent liver resection for metastases from uveal melanoma. METHODS: Over a 9-year period, patients referred with uveal melanoma metastases were included. Following treatment of primary uveal melanoma, high-risk patients were offered to be enrolled into a 6-monthly non-contrast liver magnetic resonance imaging (MRI) surveillance. Following detection of liver metastases, patients were staged with a contrast-enhanced (Primovist(®)) liver MRI, computer tomography (CT) of the thorax and staging laparoscopy. RESULTS: 155 patients were referred with uveal melanoma liver metastases, of which 17 (11.0%) patients had liver resection and one patient was treated with percutaneous radio-frequency ablation. The majority of patients undergoing liver resection were treated with multiple metastectomies (n = 8) and three patients had major liver resections. The overall median survival for patients treated with surgery/ablation was 27 (14-90) months, and this was significantly better compared to patients treated palliatively [median = 8(1-30) months, P < 0.001]. Following surgery, 11 patients had recurrent disease [median = 13(6-36) months]. Patients who had undergone a major liver resection had a significantly poorer disease-free survival (P = 0.037). CONCLUSIONS: Patients who can undergo surgical resection for metastatic uveal melanoma have a more favorable survival compared to those who do not.

Ciliary body melanoma with partial deletion of chromosome 3 detected with multiplex ligation-dependent probe amplification.

BACKGROUND: The management of a patient with a small, pigmented ciliary body tumour is controversial, the various options including observation, radiotherapy, fine needle aspiration biopsy and excision biopsy. We report a case of ciliary body melanoma with partial deletion of chromosome 3, which was missed with fluorescence in situ hybridization (FISH) but detected with multiplex ligation-dependent probe amplification (MLPA). METHODS: A 23-year-old woman underwent an excision biopsy for a 4.5 mm by 3.9 mm by 2.0 mm ciliary body tumour, which was assessed by histology, immunohistochemistry, FISH and MLPA. RESULTS: Histology showed the tumour to a melanoma of mixed cell type. FISH with a centromeric probe indicated that the tumour was of disomy 3 type with a good prognosis; however, MLPA revealed a partial deletion of the long arm of chromosome 3. CONCLUSION: This case highlights the limitations of FISH, and demonstrates the value of MLPA in testing multiple chromosomal loci of prognostic significance. To our knowledge this is the first reported case of ciliary body melanoma assessed by MLPA.

[Primary vitreoretinal lymphoma]. / Primäre vitreoretinale Lymphome.

Primary vitreoretinal lymphoma (PVRL) is the most common intraocular lymphoma. It is a high grade malignant B­cell lymphoma, which is thought to arise in the retina. It often infiltrates the central nervous system (CNS) and is therefore associated with a poor prognosis. The PVRL must be differentiated from other forms of intraocular lymphoma, such as the low-grade B­cell lymphoma that rarely arises in the choroid. The choroidal lymphomas do not spread to the brain, they can be treated with low-dose external beam radiotherapy and the patients have a good prognosis. Since PVRL is a relatively rare tumor, there is little information with respect to its true incidence, to any geographical or ethnic variability and to the main risk factors apart from an association with immunosuppression, as a result of HIV or Epstein-Barr virus infections. The treatment of PVRL is very variable between oncology centres and is also dependent on the unilaterality or bilaterality of disease and whether there is any concomitant CNS involvement. Further studies and research projects in this field are necessary in order to diagnose PVRL at an early stage and to develop new targeted individualized treatment.

Altered Nuclear Expression of the Deubiquitylase BAP1 Cannot be Used as a Prognostic Marker for Canine Melanoma.

BRCA1-associated protein-1 (BAP1) is a nuclear localized deubiquitylating enzyme that belongs to the ubiquitin c-terminal hydrolase subfamily. The encoded protein is highly homologous between man and dogs, suggesting a functional significance preserved by evolution. BAP1 has multiple properties, including tumour suppressor activity. Loss of BAP1 function is implicated in the oncogenesis of several types of cancers including uveal, mucosal and some cutaneous melanomas in humans, as well as in mesothelioma. In this study we investigate the significance of BAP1 in canine melanoma. Nuclear BAP1 protein was detected in five canine oral melanoma cell lines using an antibody commonly used for analysis of human tissues. BAP1 loss of function mutations often lead to loss of nuclear BAP1 (nBAP1) expression in humans; this is associated with a poorer prognosis in uveal and mucosal melanoma. Therefore, as a prelude to a study evaluating the prognostic significance of nBAP1 expression in dogs, immunohistochemistry (IHC) was used to assess cases of canine melanoma for nBAP1 expression. In 89 cases where tumour cells were identified by melan-A labelling, 100% of tumour cells were positive for nBAP1 expression, including eight uveal tract and 29 oral mucosal melanomas. This finding indicates that BAP1 IHC cannot be used as a prognostic marker in canine uveal and mucosal melanoma. Moreover, this observation suggests that either BAP1 has a different functional significance in canine melanoma or that loss of BAP1 function is achieved by a different route. This is a novel finding that warrants further investigation to determine the comparative biological relevance.

Primary lymphoma of the lacrimal sac: an EORTC ophthalmic oncology task force study.

AIM: To define the clinical and histopathological characteristics of primary lacrimal sac lymphoma in a predominantly white population. METHODS: Specimens of lacrimal sac lymphoma and follow up data were solicited from members of the Ophthalmic Oncology Task Force of the European Organization for Research and Treatment of Cancer (EORTC) and the European Ophthalmic Pathology Society (EOPS). Specimens were stained with haematoxylin and eosin and an immunohistochemical panel against leucocyte antigens was applied. Diagnosis was reached by consensus of five experienced pathologists according to the World Health Organization classification system. The histopathological findings were correlated with the clinical data. RESULTS: Of 15 primary lacrimal sac lymphomas, five (33%) were diffuse large B cell lymphoma (DLBCL), five (33%) were extranodal marginal zone B cell lymphoma of mucosa associated lymphoid tissue (MALT lymphoma), three were classified as "transitional MALT lymphoma," being in transition from MALT lymphoma to DLBCL, and two were unclassified B cell lymphomas. Nine of the patients were female, and the median age at the time of diagnosis was 71 years (range 45-95 years). The most frequent presenting symptoms were epiphora (85%), swelling in the region of the lacrimal sac (79%), and dacryocystitis (21%). All but one patient presented in stage I. Systemic spread occurred in three of nine patients (33%). The 5 year overall survival was 65%. CONCLUSIONS: DLBCL and MALT lymphoma are equally common in the lacrimal sac in contrast with the remaining periorbital and/or orbital region where MALT lymphoma predominates.

[Unusual masquerade of an ocular carcinoma metastasis]. / Untypische Masquerade einer okulären Karzinommetastase.

BACKGROUND: Idiopathic and therapy resistant uveitis especially of unclear origin, is a diagnostic challenge for ophthalmologists. Metastases to the anterior chamber or vitreous body can occasionally mimic the clinical picture of uveitis, a variant on the usual lymphomatous masquerade syndrome. The underlying pathological pathways leading to the metastatic spread of tumor cells within the fluid compartments of the eye remain unclear. CASE REPORT: We present an unusual case of vitreous metastases to the right eye of a patient in whom an underlying primary malignancy was unknown. After recurrent episodes of cortisone-refractive panuveitis with pseudohypopyon, a diagnostic vitreous biopsy was performed. Cytopathological examination of the vitreous sample revealed carcinoma cells with an immune profile suggestive of lung cancer metastasis. Subsequent staging investigations revealed a primary lung adenocarcinoma as well as cerebral, adrenal and osseous metastases. THERAPY: Due to the extent of dissemination of this non-small cell lung cancer (NSCLC), only palliative treatment including external beam irradiation and systemic chemotherapy was possible to reduce pain and to maintain vision as well as an attempt at systemic control of the disease.

Plasmacellular differentiation in extranodal marginal zone B cell lymphomas of the ocular adnexa: an analysis of the neoplastic plasma cell phenotype and its prognostic significance in 136 cases.

AIM: To determine (a) the expression of plasma cell related antigens in extranodal marginal zone B cell lymphomas (EMZL) of the ocular adnexa; and (b) the prognostic value of plasmacellular differentiation in these tumours. METHODS: A consecutive case series of 136 ocular adnexal EMZL obtained from three ocular pathology centres over 20 years was analysed retrospectively. An extensive immunohistochemical panel, including the plasma cell related antigens VS38c, CD38, CD138, multiple myeloma oncogene-1-protein (MUM1/IRF4), and CREB binding protein (CBP) was performed. EMZL were defined as "plasmacellular differentiated" on the basis of morphological features, evidence of cytoplasmic immunoglobulin, negativity for BSAP/PAX5, and expression of at least one of the investigated plasma cell related antigens. Controls included normal or hyperplastic lymphatic tissues. Detailed clinical data were collected for most patients, and compared with the results of immunohistochemistry. The end points considered for statistical analysis were development of local tumour recurrence, development of systemic disease, and lymphoma related death. RESULTS: 57 (42%) of the 136 ocular adnexal EMZL showed a plasmacellular differentiation; 45 of these plasmacytoid cases were primary tumours. In contrast with most admixed normal plasma cells, which displayed co-expression of MUM1/IRF4, Vs38c, CD38, CD138, and CBP, the plasmacellular differentiated EMZL tumour cells demonstrated co-expression of all five plasma cell related antigens in only six of 57 (11%) plasmacellular differentiated ocular adnexal EMZL. The most commonly expressed plasma cell related antigen was MUM1/IRF4, immunoreactivity being seen in 56/57 (98%) plasmacellular differentiated EMZL examined. Although the association of plasmacellular differentiation in primary ocular adnexal EMZL and disseminated disease was statistically significant on univariate analysis (p = 0.042), this was weaker on multivariate analysis. CONCLUSION: Plasmacellular differentiated tumour cells in EMZL demonstrate an aberrant immune profile for plasma cell related antigens when compared with normal plasma cells. On multivariate analysis, plasmacellular differentiation in ocular adnexal EMZL was not significantly associated with local recurrence, the development of systemic disease, or with lymphoma related death.

[Characterization of an experimental venous thrombus model with MRI, phlebography and histology]. / Charakterisierung eines experimentellen venösen Thrombosemodells mittels MRT, Phlebographie und Histologie.

INTRODUCTION: Several magnetic resonance (MR) techniques designed to demonstrate the characteristic signal intensity of blood degeneration products of thrombi have been suggested, but the effect of thrombus organization on the MR display, in particular with regard to its temporal evolution, remains to be determined. It is the purpose of this study to develop a stagnation thrombus model in rabbits and to characterize thrombus at different ages with two (MR) imaging techniques, phlebography and histology. MATERIALS AND METHODS: Venous stagnation thrombi were induced in the external jugular veins of rabbits using a minimally invasive radiological technique to produce artificial embolic vascular occlusion and hypercoagulability. Twenty-five animals were divided into 5 groups of 5 animals, and each group underwent 1.5 T MR imaging at 1, 3, 5, 7 and 9 days after thrombus induction using a T1-weighted magnetization-prepared rapid gradient-echo sequence (MP-RAGE: TR 10.4 msec, TE 4.0 msec, FA 15 degrees ) and a T2-weighted fast low-angle shot sequence (FLASH: TR 54 msec, TE 18 msec, FA 15 degrees ). The thrombus length was measured on the T1-weighted images. Thrombus conspicuity, signal intensity, and heterogeneity on T2* weighted images were described using visual scales. Radiographic venography and histology served as reference methods. RESULTS: Thrombi were successfully induced in all animals. The overall thrombus length decreased from 43 +/- 9 (day 1 after induction) to 23 +/- 4 mm (day 9). On 3D-reconstructions of the T1-weighted images, the visible portion of the true thrombus length relative to the overall thrombus length was 0.16 +/- 0.3 (day 1), 0.24 +/- 0.3 (day 3), 0.38 +/- 0.5 (day 5), 0.06 +/- 0.1 (day 7) and 0.00 (day 9). Sixteen of 25 thrombi were detectable with the T2*-weighted technique. The overall thrombus signal intensity decreased with the age of the thrombus from day 1 to day 9. The histological evaluation showed that the rabbit thrombi closely resemble human thrombi morphologically. CONCLUSIONS: The thrombus model closely resembles the human venous stagnation thrombus of different organizational stages. With state-of-the-art MRI techniques, thrombi were only partially displayed with the visibility depending on thrombus age. The model may be suitable for evaluating new and potentially more effective MRI techniques for improved thrombus visualization.

Immunoglobulin VH gene expression among extranodal marginal zone B-cell lymphomas of the ocular adnexa.

PURPOSE: Most lymphomas of the ocular adnexa are primary extranodal non-Hodgkin's lymphomas of the B-cell type, with the most common lymphoma subtype being the extranodal marginal-zone B-cell lymphoma (EMZL). Analysis of somatic mutations in the variable (V) region of the Ig heavy (H)-chain gene segment suggests that EMZL development in other locations is dependent on antigen stimulation. The purpose of this study was to analyze the presence of somatic hypermutations in clonally rearranged Ig H-chain V genes of this lymphoma entity in the ocular adnexa and to estimate whether the mutation pattern is compatible with antigen selection. METHODS: Twenty-six cases of EMZL of the ocular adnexa were diagnosed on the basis of morphology, histology, and immunohistology. A nested polymerase chain reaction (PCR) was performed on DNA extracted from paraffin sections. The isolated PCR products were sequenced and compared with published VH germline segments to determine the number of somatic mutations in the complementarity-determining region (CDR) 2 and framework (FW) region 3. RESULTS: The number of somatic mutations in the cases of EMZL varied between 0 and 24: Five cases involved 0 to 3 somatic mutations, and the remaining 21 cases involved 4 to 24 mutations. Based on the ratio of replacement (R) to silent (S) mutations in the CDR2 or FW3 regions, antigen selection seems to have occurred in 60% of ocular adnexal EMZL. The VH3 family was the most commonly expressed germline VH family (54%), followed by VH4 (23%), with biased usage of the latter. Some germline VH1 genes used included DP-8, DP-10, DP-53, DP-63 (VH4.21), and DP-49, which are frequently used by autoantibodies (e.g., rheumatoid factors) and natural autoantibodies. CONCLUSIONS: EMZLs of the ocular adnexa have an Ig H-chain mutation pattern that supports the concept that they represent a clonal expansion of post-germinal-center memory B-cells in most instances. In two thirds of cases, antigen selection may have occurred, and autoantibodies may have a role in their development.

Superparamagnetic iron oxide-enhanced MRI of atherosclerotic plaques in Watanabe hereditable hyperlipidemic rabbits.

RATIONALE AND OBJECTIVES: Inflammatory atherosclerotic plaques are characterized by increased endothelial permeability and multiple macrophages. Blood-pool MRI contrast agents like superparamagnetic iron oxide (SPIO) have an affinity for the monocyte-macrophage system and thus, may label inflammatory plaques. The objective was to demonstrate SPIO uptake in plaques of atherosclerotic rabbits by MRI and histology. METHODS: Aortas of anesthetized Watanabe hereditable hyperlipidemic rabbits were studied with a moderately T2*-weighted gradient-echo sequence at 1.5 T. Four groups of five animals each were studied: (1) without ultrasmall SPIO (carboxydextran coating; particle size, 25 nm; estimated plasma half-life, 6 hours) or with imaging after intravenous injection of SPIO at a dose (micromol Fe/kg) and postcontrast time delay (hours) of 50/8 (2), 50/24 (3), or 200/48 (4). In vivo MRI was compared with corresponding ex vivo histological iron stains. RESULTS: Animals receiving 200 micromol Fe/kg demonstrated areas of focal signal loss clearly confined to the aortic wall on a mean of 24 +/- 9 (31% +/- 11%) of 76 +/- 5 images compared with 0 +/- 0 of 76 +/- 5 images in controls (P = 0.009). The number of images with this finding in groups 2 and 3 was not significantly different compared with controls. By microscopy, SPIO-iron was seen in the endothelial cells and subendothelial intimal macrophages of atherosclerosis-prone aortic wall segments. Atherosclerotic lesions demonstrating iron uptake also showed a high macrophage content. CONCLUSIONS: SPIO accumulates in aortic plaques of atherosclerotic rabbits, producing a characteristic MRI finding. As SPIO accumulates in plaques with increased endothelial permeability and a high macrophage content, two established features of plaque inflammation, it may have a potential for noninvasive assessment of inflammatory atherosclerotic plaques.

Iron-oxide-enhanced magnetic resonance imaging of atherosclerotic plaques: postmortem analysis of accuracy, inter-observer agreement, and pitfalls.

INTRODUCTION: Contrast-enhanced magnetic resonance (MR) imaging using ultra small superparamagnetic iron oxide (USPIO) particles is a new noninvasive modality for imaging inflammatory atherosclerotic plaques. We determined the accuracy, interobserver agreement, and potential sources of error of this technique by means of postmortem MR imaging of aortic preparations. MATERIAL AND METHODS: Anesthetized atherosclerotic Watanabe heritable hyperlipidemic (WHHL) rabbits were studied after administration of different dosages of intravenous USPIO (DDM 43/34, IDF Berlin, Germany) and different postcontrast time intervals. A (n = 5) received 0 micromol Fe/kg. B (n = 5) received 50 micromol Fe/kg, 8-hour postcontrast interval. C (n = 5) received 50 micromol, 24 hours. D received 200 micromol, 48 hours. The aortas were removed and 3-mm segments prepared for postmortem examination by MR imaging using a T2-weighted gradient-echo sequence (TR/TE/FA; 41 milliseconds/11 milliseconds/15 degrees ), radiography (mammography), and histology (iron staining). USPIO accumulation was defined as the presence of 20 iron-positive cells per microscopic view (x100 magnification). Two independent readers analyzed the MR images and rated their confidence level for a positive MRI finding, defined as a focal signal loss, on a 5-point scale. The results were evaluated by receiver-operator characteristic (ROC) analysis. RESULTS: Of a total of 621 vessel segments technically acceptable for evaluation, 534 were histologically negative and 87 positive. Accuracy, expressed as the area under the ROC curve, was 0.85 for reader 1 and 0.88 for reader 2. Interobserver agreement was 0.67. False-positive findings were established by at least one reader for 121 of the 621 segments, false-negative findings for only 15 segments. Calcifications and mural thrombi were identified as potential sources of error of the method. CONCLUSION: Postmortem USPIO-enhanced MR imaging of atherosclerotic plaques showed a high accuracy and good interobserver agreement in the animal model used here. Further optimization of the method should aim at reducing the rather high percentage of false-positive results.

Clinicopathologic features of retinoblastoma after primary chemoreduction.

BACKGROUND: Primary chemotherapy is a new treatment approach in retinoblastoma, aiming to avoid radiogenic adverse effects, such as second tumor-associated mortality, as observed following external beam irradiation. OBJECTIVE: To describe the clinical and histopathologic regression pattern after primary chemotherapy in retinoblastoma. METHODS: Five patients with sporadic bilateral retinoblastoma underwent planned enucleation of their functionally blind eye after 2, 3 (in 2 patients), 4, and 6 courses of primary chemotherapy with carboplatin, etoposide, cyclophosphamide, and vincristine. The eyes were examined histopathologically, using light microscopy and immunohistochemical analysis with proliferation markers. RESULTS: One patient had a type 1 (cottage cheese) regression and 4 patients had either a type 2 (fish flesh) or a type 3 (combined) regression pattern. Histopathologic examination revealed a complete tumor necrosis in 1 patient with type 1 regression after 3 courses of chemotherapy and in 1 patient with type 3 regression after 4 courses of chemotherapy. The remaining 3 patients with type 2 or type 3 regression had histologically still active proliferative tumor cells after 2, 3, and 6 courses of chemotherapy. CONCLUSION: This article correlates histopathologically the clinically described efficacy of primary chemotherapy in the treatment of retinoblastoma, underlining, however, the necessity of careful observation and the use of ancillary treatment whenever there is no complete tumor regression.