Expression of cancer-testis antigens (MAGE-A1, MAGE-A3/6, MAGE-A4, MAGE-C1 and NY-ESO-1) in primary human uveal and conjunctival melanoma.

AIM: Metastatic disease in ocular melanoma remains untreatable, is associated with late detection and is resistant to conventional systemic therapies. Many tumours including cutaneous melanoma express specific cancer-testis (CT) antigens and vaccines targeting these antigens can induce T-cell-mediated and humoural immune responses. The authors examined primary uveal and conjunctival melanomas for expression of CT antigens to assess their potential as targets for ocular melanoma immunotherapy. METHODS: Paraffin-embedded uveal (n=32) and conjunctival (n=15) melanomas were assessed by immunohistochemistry for melanocyte differentiation antigens (gp100, Melan-A/MART-1 and tyrosinase), and CT antigens (MAGE-A1, MAGE-A3/6, MAGE-A4, MAGE-C1 and NY-ESO-1). RESULTS: Melanoma differentiation antigens, gp100, Melan-A/MART1 and tyrosinase, were expressed in >75% of tumour cells in all uveal and conjunctival melanomas tested. Expression of all five CT antigens tested was low in uveal melanomas, and when present, stained <25% of the tumour cells. MAGE-A1, MAGE-A4 and NY-ESO-1 were expressed in <10% of tumour cells in conjunctival melanomas, while MAGE-C1 and MAGE-A3/6 were expressed in ∼20% and ∼35% of tumour cells in this malignancy, respectively, with variable expression levels. CONCLUSIONS: Uveal and conjunctival melanomas consistently expressed high levels of the differentiation antigens (gp100, Melan-A/MART1 and tyrosinase). However, compared with other tumours, including cutaneous melanoma, only low levels of CT antigens were found in ocular melanomas. These observations suggest that immunotherapy directly targeting the CT antigens studied may not be effective for ocular melanoma.

The role of a multidisciplinary pre-assessment clinic in reducing mortality after complex orthopaedic surgery.

INTRODUCTION: This paper describes an audit loop. The aim of this study was to audit the effect of a specialised preoperative anaesthetic assessment clinic after hip and knee arthroplasty and revision arthroplasty. PATIENTS AND METHODS: We studied patients undergoing hip and knee surgery (arthroplasty and revision arthroplasty). We collected data concerning postoperative admissions to the high dependency unit (HDU), intensive care unit (ICU) and post-anaesthesia care unit (PACU) (planned and unplanned rates of admission, length of stay). We also noted mortality. In the first part of the study (April 2005 to March 2006) we studied 298 patients. All patients were assessed independently by an anaesthetist on the day of surgery. A multidisciplinary preoperative assessment clinic commenced in April 2006. After this date all patients were assessed preoperatively by a multidisciplinary anaesthetic lead team. In the second part of the study (May 2006 to April 2009) a further 1,147 arthroplasty patients were studied. Data were again collected regarding HDU, ICU, PACU and mortality, as noted above. RESULTS: We found statistically significant (p = 0.001) reductions in the admissions to PACU (22% down to 10%) and in mortality (6.1% down to 1.2%) after the introduction of the pre-assessment clinic. There was also a statistically significant (p = 0.01) reduction in the HDU length of stay (2.1 days to 1.6 days), ICU unplanned admissions (1.3% to 0.4%) and the ICU length of stay (2.3 to 1.9 days). We estimated cost savings of nearly £50,000 in the second part of the study. This is based on the average decrease in HDU and ICU length of stay. CONCLUSIONS: We recommend the use of a multidisciplinary pre-assessment clinic for complex orthopaedic surgery.

Expression and distribution of MMPs and TIMPs in human uveal melanoma.

Matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) are involved in tumour invasion, metastasis and angiogenesis, and have been implicated as progression markers in uveal melanoma, although their topographical expression has not been fully described. In this study we compared the distribution and specificity of several classes of MMPs (MMP-1, -2, -9, -19, and MT1-MMP) and physiological MMP inhibitors (TIMP-2 and -3) in different regions of the tumour microenvironment and adjacent choroid in a series of primary uveal melanomas. Paraffin sections of untreated uveal melanomas (n=18, 3/18 spindle; 11/18 mixed, and 4/18 epithelioid) were examined for MMP-1 (collagenase 1), MMP-2 and MMP-9 (gelatinases A and B), MT1-MMP (membrane-type 1-MMP), MMP-19, TIMP-2 and TIMP-3 (tissue inhibitors of MMPs), using indirect peroxidase immunohistochemistry. The distribution and intensity of immunolabelling was graded semi-quantitatively (0-3) by 2 independent observers. Non-parametric analyses were used to test for associations between tumour cell type, and the average grade of MMP or TIMP expression. Immunostaining for MMP-1, -9, -19 and MT1-MMP was > or =Grade 2 in more than 70% of specimens, and a heterogeneous pattern of MMP-1, -9, MT1-MMP and TIMP-3 expression was observed. At the tumour-scleral interface (TSI), melanoma cells had a flattened morphology and a much reduced MMP and TIMP expression, with a high expression in tumour areas adjacent to the TSI. Tumour vasculature and stromal cells strongly expressed MMP-2. We also observed heterogeneous immunostaining of the vasculature by MMP-1, -9, MT1-MMP and TIMP-2 antibodies, and of the extravascular matrix by MMP-9 antibody. The distinct immunostaining patterns observed for MMPs and TIMPs within uveal melanoma are consistent with their involvement in tumour growth and angiogenesis. In particular, the heterogeneous expression within regions of the tumours, and the localized expression in vasculature and stromal cells emphasises the importance of the tumour microenvironment in the pathogenesis of uveal melanoma (and other tumours).

Beta-lapachone inhibits proliferation and induces apoptosis in retinoblastoma cell lines.

AIMS: To investigate the cytotoxicity of beta-lapachone, a potent agent that may selectively target tumour cells, in retinoblastoma (RB) cell lines. METHODS: Growth inhibitory effects of beta-lapachone were evaluated in Y79, WERI-RB1, and RBM human retinoblastoma cell lines. Pro-apoptotic effects of beta-lapachone were evaluated in Y79 cells by detection of caspase 3/7 activity, by enzyme-linked immunosorbent assay for nucleosome fragments, and by cellular morphological analysis. RESULTS: Beta-lapachone induced significant dose-dependent growth inhibitory effects in all three retinoblastoma cell lines. The 50% growth inhibitory concentration (IC(50)) of this agent was 1.9 microM in Y79 cells, 1.3 microM in WERI-RB1 cells, and 0.9 microM in RBM cells. Beta-lapachone also induced proapoptotic effects in RB cells. Treatment of Y79 cells with 1.9 microM beta-lapachone (IC(50)) resulted in a peak, fourfold induction of caspase 3/7 activity at 72 h post-treatment; a peak, 5.6-fold increase in nucleosome fragments at 96 h post-treatment; and a peak, 1.7-fold increase in the frequency of apoptotic cells at 48 h post-treatment, relative to vehicle-treated controls. CONCLUSION: Beta-lapachone induced potent cytotoxic effects in RB cell lines at low micromolar concentrations, suggesting this agent could be useful in the clinical management of RB.

Ultrasound biomicroscopy: role in diagnosis and management in 130 consecutive patients evaluated for anterior segment tumours.

BACKGROUND/AIM: Ultrasound biomicroscopy (UBM) is an important tool for assessing anterior segment pathology. This study sought to evaluate UBM in the management of anterior segment tumours. METHODS: Retrospective analysis of medical records of consecutive patients referred to the ocular oncology unit, University of California San Francisco (UCSF), for suspected anterior segment tumours from 1999 to 2004. RESULTS: 132 eyes from 130 patients were evaluated, including 55 uveal melanomas (UM), 21 iris naevi, 30 iris cysts, and 26 remaining lesions. Of the melanomas, 45 were also evaluated with conventional A/B-scan. There was 29% correspondence between the anatomical structures invaded by melanoma as identified by B-scan v disease extent defined by UBM. Ciliary body and peripheral iris involvement by melanomas was significantly more frequently observed by UBM than B-scan. Seven of 30 benign cysts were diagnosed as cystic before UBM evaluation. In three cases, neuroepithelial cysts were associated with intercurrent pathology including iris naevus (n = 2) and ciliary body melanoma (n = 1). Two ciliary body melanomas showed cavitation, including one patient with a pseudocyst. Histopathological correlation was possible in six cases. CONCLUSION: UBM is an indispensable tool for the management of anterior segment tumours. This study demonstrates the superiority of UBM v conventional B-scan for the precise localisation of uveal melanoma, especially involving the ciliary body and peripheral iris.

Surgery for primary basal cell carcinoma including the eyelid margins with intraoperative frozen section control: comparative interventional study with a minimum clinical follow up of 5 years.

AIM: To assess recurrence of primary basal cell carcinoma (PBCC) including the eyelid margins after resection with or without intraoperative frozen section control (IFS). METHODS: Comparative non-randomised interventional study involving review of records of consecutive patients with histological diagnosis of PBCC including the eyelid margins, treated surgically at the University of Erlangen-Nürnberg between 1989 and 1998. Patients with a minimum clinical follow up of 5 years treated with (group I) or without (group II) IFS were compared. Postoperatively, permanent paraffin sections were available in all patients. RESULTS: 165 patients were available for study. There were 145 patients with a minimum of 5 years follow up. Of these, no tumour recurrences were observed in group I (n = 114) compared with three (9.7%) in group II (n = 31) (p = 0.002). CONCLUSIONS: Surgery for PBCC including the eyelid margins with IFS and immediate plastic reconstruction, as performed in the present study, is associated with better long term cure compared with surgery with clinical control.

[Management of conjunctival malignant melanoma associated with primary acquired melanosis (PAM) using 0.02% mitomycin C eyedrops]. / Therapie maligner Melanome der Konjunktiva bei primär erworbener Melanose (PEM) mit Mitomycin-C-0,02%-Augentropfen.

BACKGROUND: The therapy of malignant diseases of the conjunctiva with local chemotherapy is an extension of the therapeutic options in this field. We report on our experience in the therapy of malignant melanomas of the conjunctiva associated with primary acquired melanosis (PAM). METHODS AND PATIENTS: Between March 1998 and April 2001, 13 patients with malignant melanoma of the conjunctiva associated with PAM (6 female; 7 male; mean age 57+/-13 years) were treated with local chemotherapy. The tumor was classified as stage pT2 (pN0, pM0) in seven patients, stage pT3 (pN0, pM0) in three patients and in the remaining three patients the lid was involved in the malignant process (pT4, pN0, pM0). Local chemotherapy (mitomycin C 0.02% eyedrops 5 times a day) was applied after incisional biopsies in 2 cycles for 14 days with a 14-day break. In 4 patients a third cycle was included. RESULTS: Regression of the tumor was observed after completion of the therapy in all cases. Severe ocular or systemic secondary effects were not seen in our patients. Nine patients were without recurrence within the follow-up time. In three patients, a recurrence of the disease was observed. In these cases, the eyelid was involved in the process. CONCLUSION: Local chemotherapy with mitomycin C is a useful option in the treatment of malignant melanomas of the conjunctiva associated with PAM if the tumor stage is pT3 or less. From our point of view the combination with incisional biopsy is of great benefit. Prognosis of conjunctival malignant melanomas involving the lid margin (pT4) is poor.

Human uveal melanoma expresses NG2 immunoreactivity.

BACKGROUND/AIMS: NG2 is the rat homologue of the human melanoma proteoglycan (HMP), also known as the high molecular weight melanoma associated antigen. Most cutaneous melanomas, as well as glioblastomas, chondrosarcomas, and some leukaemias express NG2 immunoreactivity, recognised using monoclonal antibody (mAb) 9.2.27. This antibody has also been used for molecular targeting in targeted alpha therapy for melanoma. The purpose of this study was to evaluate the expression of NG2 immunoreactivity in human uveal melanoma and normal ocular tissue using mAb 9.2.27. METHODS: Enucleated eyes from 26 patients with choroidal or ciliary body melanoma (n=26) were available as paraffin sections, and stained with haematoxylin and eosin to assess for tumour cell type and histopathology. Additional slides were investigated for NG2 immunoreactivity using mAb 9.2.27 and alkaline phosphatase anti-alkaline phosphatase (APAAP) immunostaining. Two independent observers graded immunostaining using a semiquantitative scale from 0 (negative) to 3 (strong). RESULTS: Immunostaining for mAb 9.2.27 could not be graded in 7/26 cases with dense pigmentation of the tumour. For the remaining cases, grade 2 (moderate) or more immunostaining was seen in 18/19 tumours (95%). The retina, retinal pigment epithelium (RPE), and choroid displayed weak immunostaining (grade 0.5-1.5) in the majority of melanoma affected eyes. Normal retina and choroid (n=5) appeared negative for mAb 9.2.27. Optic nerve axon bundles in both control and melanoma affected eyes displayed moderate immunostaining. CONCLUSION: In the present study, the majority of human uveal melanomas expressed NG2 immunoreactivity, as detected using mAb 9.2.27. This antibody may be a suitable candidate for radioimmunotherapy to target ocular melanoma.

Biomolecular markers of malignancy in human uveal melanoma: the role of the cadherin-catenin complex and gene expression profiling.

In recent years there has been a trend towards conservative management of uveal melanoma (UM), aimed at preserving the eye and vision. Despite improvements with this approach, recurrent tumour and metastatic disease still occur, and the management remains problematic. As a result of these limitations, there is interest in gaining a greater understanding of molecular changes associated with aggressive disease patterns in UM. This might result in new, more effective and less toxic therapies as well as provide prognostic information for defining subgroups of patients with a less favourable prognosis as potential candidates for adjuvant therapies. Accumulating evidence over the past decade suggests that disturbance in the cadherin-catenin adhesion complex is critical in the process leading to invasion and metastasis of many cancers. The recent advent of DNA micro-array technology now offers an unprecedented ability to study these molecules and others associated with malignant transformation. In this mini-review, the aspects of tumour progression in which cadherin-catenin may be involved are dealt with along with the potential application of DNA micro-array technology to the problem in UM.

Primary iris melanoma: diagnostic features and outcome of conservative surgical treatment.

AIMS: To describe features influencing the management of primary iris melanoma and report the outcome of conservative surgical treatment of patients diagnosed with this condition in a tertiary referral academic setting over a 20 year period. METHODS: Retrospective non-comparative case series of consecutive patients diagnosed with iris melanoma from 1980-2000 using medical records from the University of Sydney Department of Ophthalmology and NSW Cancer Registry RESULTS: 51 cases were identified. The most common presentation was growth of a previously noted pigmented lesion. Initial management was either observation or local resection (two had enucleations) with iris reconstruction where possible (23.8%). The mean follow up was 8.7 years (range 1-17 years). Vision of 6/12 or better was maintained in the majority (78.6%) treated by local resection. Pupil reconstruction significantly reduced reported postoperative glare symptoms. Four patients had features suggestive of local recurrence and there was no documented metastatic disease or death from iris melanoma in this series. Histologically, the majority were spindle B cell melanomas. Clinical features including prominent tumour vascularity, rapid growth, and heterogeneous pigmentation were each significantly associated with an epithelioid cell component. Involvement of the iridocorneal angle was frequently associated with ciliary body invasion. CONCLUSIONS: Management decisions for iris melanoma will depend on the clinical features. Mixed or epithelioid histology is more likely in the presence of two or more of the features of malignancy and may justify earlier intervention. When treatment is undertaken, local resection achieves long term tumour clearance with an acceptable morbidity. In resecting iris melanoma, careful assessment for iridocorneal angle involvement is important in treatment planning. Iris reconstruction has a useful role in reducing postoperative photophobia.

The development of ISO 9002 quality management standards for Canadian dental practices.

The department of dentistry of the Hospital for Sick Children has actively maintained a quality assurance system since the early 1980s. In addition, members of the department have taught courses and published articles on risk management and quality assurance for over a decade. The decision to achieve ISO 9002 registration led to an intensive 10-month process to adapt ISO systems and standards to Canadian institutional dental practice. This article describes the ISO registration system and the changes required for an existing quality assurance program to conform to ISO standards.

Sodium butyrate modulates p53 and Bcl-2 expression in human retinoblastoma cell lines.

Sodium butyrate (SB) is a potent biological modifier that can induce diverse effects including growth inhibition, differentiation, or apoptosis of many cell types including retinoblastoma (Rb), and modulation of genes such as c-fos and p53. In this study we assessed the effects of SB on cell growth and expression of p53, critical for cell cycle control, and Bcl-2, an inhibitor of apoptosis, in two human Rb cell lines (Y79 and WERI-Rb1). Attachment cultures were treated with 1 mM SB for up to 5 days and immunocytochemistry was used to examine for the expression of neural cell adhesion molecule (NCAM), p53, and Bcl-2. Suspension cultures of both cell lines were also treated with 1 and 4 mM SB, and at selected times cell extracts were prepared and the expression of p53 and Bcl-2 proteins determined by Western blot analysis. Treatment with 1 mM SB of both cell lines for 5 days inhibited growth and induced morphological changes including extension of neurite-like processes. Up to 12 h after 1 mM SB treatment, p53 and Bcl-2 expressions were similar to control levels, then gradually decreased to very low levels at 5 days. SB (4 mM) also inhibited growth associated with cell death, which was apparent at 24 h posttreatment. Expressions of p53 and Bcl-2 were decreased below control levels at 4 h, and by 24 h only very low levels of protein were detected. SB-induced modulation of p53 and Bcl-2 expression may have implications for controlling Rb growth, particularly in combination with chemotherapy drugs, which are increasingly used in the treatment of Rb.

Vincristine- and cisplatin-induced apoptosis in human retinoblastoma. Potentiation by sodium butyrate.

Chemotherapy alone has largely been unsuccessful in controlling retinoblastoma growth, and has traditionally been limited in use as an alternative to irradiation for the treatment of retinoblastoma. Recently, clinical studies combining chemotherapy with local therapies, including radiotherapy, laser therapy or cryotherapy and in some cases, cyclosporine A, have been effective in treating retinoblastoma. Differentiating agents may also be combined with chemotherapy to enhance the action of cytotoxic drugs on tumor cell growth, although this approach has not been fully investigated in retinoblastoma. In this study, we evaluated the cytotoxic response of human retinoblastoma cell lines (Y79 and WERI-Rb1) to two chemotherapy agents commonly used in treating retinoblastoma, vincristine (VCR) and cisplatin (CDDP). Retinoblastoma cells have been shown to be sensitive to the differentiating agent sodium butyrate, and cell lines were also treated with a combination of VCR or CDDP with sodium butyrate, and the effects on retinoblastoma viability assessed. Both VCR and CDDP induced dose-dependent death of Y79 and WERI-Rb1 cells, accompanied by nuclear and cytoplasmic condensation and DNA laddering, features characteristic of apoptosis. Inhibitors of macromolecular synthesis, cycloheximide and actinomycin-D, significantly reduced VCR- and CDDP-induced apoptosis, although putative endonuclease inhibitors zinc sulphate and aurintricarboxylic acid had no apparent effect. Treatment with 0.5 mM or 1 mM sodium butyrate combined with VCR or CDDP significantly increased induction of apoptosis by these agents. This augmentation of chemotherapy-induced apoptosis may have implications for retinoblastoma therapy.

Human retinoblastoma: in vitro differentiation and immunoglobulin superfamily antigen modulation by retinoic acid.

Suspension and attachment cultures of Y79 human retinoblastoma cells were treated with all-trans retinoic acid (RA) for up to 10 days to assess its effect on growth and cell-surface expression of immunoglobulin superfamily antigens MHC class I and class II, ICAM-1, NCAM and Thy1. RA up to 10 microM induced growth inhibition, and marked morphological differentiation with extension of prominent processes resembling neurites was seen in attachment cultures. However, above 10 microM RA produced extensive cell death. We also observed increased cell-surface expression of MHC class I, ICAM-1, NCAM and Thy1 on Y79 cells treated with 10 microM over 10 days; constitutive MHC class II expression was not apparent, nor did RA treatment appear to induce Y79 cells to express MHC class immunoreactivity. The up-modulation of cell-adhesion molecules (NCAM, ICAM-1 and Thy1) and immune recognition molecules (NCAM, ICAM-1 and MHC class I), associated with reduced growth and tumour cell differentiation, suggests that RA may have a potential role in regulating the growth and development of retinoblastoma tumours.

Incomplete Reiter's syndrome with focal involvement of the posterior segment.

PURPOSE: To describe an unusual variant of Yersinia-induced, HLA-B27 associated incomplete Reiter's syndrome with focal involvement of the posterior segment. METHODS: Review of case records of a patient presenting with incomplete Reiter's syndrome which included a reactive arthritis with keratoconjunctivitis and anterior uveitis. RESULTS: The uveitis progressed to involve the posterior segment with a vitritis and two transient white retinal spots. After resolving, a retinal pigment epithelial (RPE) defect persisted at the site of one of the lesions. CONCLUSIONS: While involvement of the anterior segment of the globe in Reiter's disease is well recognised, a review of the literature reveals that focal posterior involvement is a rare feature in either Reiter's syndrome or the reactive arthritis group.

Induction of apoptosis by sodium butyrate in the human Y-79 retinoblastoma cell line.

The mode of cell death induced in the Y-79 human retinoblastoma cell line by sodium butyrate (SB), a short-chain fatty acid with potent inhibitory effects on the growth of many transformed cell lines, was investigated by fluorescence and transmission electron microscopy, agarose gel electrophoresis, and metabolic studies. While SB (< 1 mM) resulted in marked morphological differentiation, higher concentrations (1-4 mM) induced predominantly apoptotic involution in Y-79 in a concentration-dependent fashion after a latent period of 24 h. Dying cells displayed the characteristic morphology of apoptosis accompanied by DNA laddering with agarose gel electrophoresis. Extensive cell necrosis was apparent with 0.5 M SB. Induction of apoptosis and DNA laddering by SB was reduced by putative inhibitors of RNA and protein synthesis, but not putative endonuclease inhibitors. These results are important for understanding the mode of action of sodium butyrate as a potential cancer chemotherapeutic agent.