Kenneth Lai

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 α 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.

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).

Gene of the month: PIK3CA

PIK3CA encodes the p110α catalytic subunit of phosphatidylinositol 3-kinase (PI3K) which through its role in the PI3K/Akt pathway is important for the regulation of important cellular functions such as proliferation, metabolism and protein synthesis, angiogenesis and apoptosis. Mutations in PIK3CA are known to be involved in a wide range of human cancers and mutant PIK3CA is thought to act as an oncogene. The specific PIK3CA inhibitor, NVP-BYL719, has displayed promising results in cancer therapy and is currently under clinical trials. Furthermore, PI3K regulates autophagy, a cellular process that recycles proteins and organelles through lysosomal degradation and has recently been recognised as an attractive therapeutic target due to its pro- and anti-cancer properties. Several studies have attempted to investigate the effects of combining the inhibition of both PI3K and autophagy in cancer therapy, and an in vivo model has demonstrated that the combined use of a concomitant PI3K and autophagy inhibitor induced apoptosis in glioma cells.

The significance of autophagy in colorectal cancer pathogenesis and implications for therapy

Colorectal cancer (CRC) is one of the most common cancers in developed countries with poor survival outcome in advanced stages of the disease due to its resistance to chemotherapy and other forms of treatment. New and alternative approaches are needed to overcome the tumour cells’ capacity for survival and to drive the tumour towards cell death. Autophagy is a mechanism involved in the elimination of damaged cellular components through lysosomal degradation and is capable of inducing programmed cell death. The process has recently gained much interest in understanding the pathogenesis of CRC and its potential for treatment of the disease due to its role in host protection and anticancer activity. This review describes and illustrates the fundamental mechanisms of autophagy, its importance as a prognostic marker and the current approaches to harness its protective and anticancer activity in CRC therapy.

Quality assessment and preservation of RNA from biobank tissue specimens: a systematic review

It is well recognised that genomic, proteomic and biomarker studies require properly annotated and well-characterised biospecimens. Consequently, this necessitates biobanks to collect, store and distribute biospecimens under stringent quality control and assurance measures. However, despite this realisation, there remains a lack of standardisation in quality management among biobanks and consensus as to which quality indicators provide the optimal molecular diagnostic performance tools and information for biospecimens. In an attempt to identify key factors that predict tissue specimen integrity and quality, this systematic review investigated the measures reported in the literature, which characterised the collection, processing and storage of high-quality tissue specimens. Our findings demonstrated RNA integrity, alone, may not be an effective measure of tissue quality. Furthermore, the frequently reported parameters related to biospecimen integrity, such as storage time, temperature, time to cryopreservation and tissue morphology were also not effective indicators of quality control and assurance. These findings suggest that it is unlikely that a single marker will provide the optimal diagnostic and performance information for biospecimens, but rather, a panel of markers assessing the molecular integrity of the lifespan of the biospecimen is required. Further work is needed to identify which factors predict specimen integrity and quality in biobanked tissue specimens.

Renal biopsy pathology in a cohort of patients from southwest Sydney with clinically diagnosed systemic lupus erythematosus

Purpose The pathological manifestations in the kidneys in systemic lupus erythematosus (SLE) are commonly known as lupus nephritis. We have studied the pathological changes in renal biopsies from 59 cases of clinically diagnosed SLE obtained over a 15-year period from a racially diverse population in the Sydney metropolitan area. Our aim was to see if there was any regional variation in the morphological changes. Methods Renal biopsy changes were assessed by routine light, immunofluorescence, and electron microscopy. We used the modified 1974 World Health Organization classification of lupus nephritis to classify cases into six classes. Disease severity was assessed by age, sex, and across racial groups, including Caucasian, Asian, Middle Eastern, Mediterranean, Indian subcontinental, South American, and Pacific Islander. Results Our analysis showed that cases of lupus nephritis contributed 5.4% of our total renal biopsies examined over a 15-year period. The overall incidence of biopsy-proven cases was 0.49 per 100,000 per year. The ages of our patients ranged from 10 to 79 years, with most below 50 years of age. A female to male ratio was determined to be 4.4:1. There was no relationship to ethnicity, nor was there a relationship between any of these parameters and the class or severity of disease. Conclusion Renal biopsy with multimodal morphological and immunohistochemical analysis remains the gold standard for diagnosis and determination of the level of disease in lupus nephritis. Based on this approach we have identified an incidence rate for southwest Sydney that is slightly higher but comparable to that found in a similar study from the United Kingdom. We also found that there was no relationship between sex, race, or age and severity of disease.

Predicting the response of multiple myeloma to the proteasome inhibitor Bortezomib by evaluation of the unfolded protein response

Predicting the response of multiple myeloma to the proteasome inhibitor Bortezomib by evaluation of the unfolded protein response

Quantum Dot Immunocytochemical Localization of Somatostatin in Somatostatinoma by Widefield Epifluorescence, Super-resolution Light, and Immunoelectron Microscopy

Quantum dot nanocrystal probes (QDs) have been used for detection of somatostatin hormone in secretory granules of somatostatinoma tumor cells by immunofluorescence light microscopy, super-resolution light microscopy, and immunoelectron microscopy. Immunostaining for all modalities was done using sections taken from an epoxy resin-embedded tissue specimen and a similar labeling protocol. This approach allowed assessment of labeling at light microscopy level before examination at super-resolution and electron microscopy level and was a significant aid in interpretation. Etching of ultrathin sections with saturated sodium metaperiodate was a critical step presumably able to retrieve some tissue antigenicity masked by processing in epoxy resin. Immunofluorescence microscopy of QD-immunolabeled sections showed somatostatin hormone localization in cytoplasmic granules. Some variable staining of tumor gland-like structures appeared related to granule maturity and dispersal of granule contents within the tumor cell cytoplasm. Super-resolution light microscopy demonstrated localization of somatostatin within individual secretory granules to be heterogeneous, and this staining pattern was confirmed by immunoelectron microscopy.

21. The emerging role of nanotechnology in anatomical pathology

Background: Interpretation of subcellular localisation by immunoelectron microscopy is often difficult due to low probe density on target structures. Correlation studies can provide more accurate context and spatial resolution but such approach has hindered by a lack of suitable probes, furthermore, conventional light microscopy will also limit the interpretation due to the resolution limit from diffraction. Nanoparticles such as quantum dots are recently being introduced in research applications. These nanocrystals offer the potential of universal markers and can be visualised by light and electron microscopy. Super-resolution light microscopy overcomes the limit of light diffraction hence it allows a higher image resolution than the conventional light microscopy. Aims: To investigate quantum dot for use as immunocytochemical probes for correlation between light and electron microscopy. Furthermore the image quality between super-resolution, confocal and fluorescence light microscopy were also assessed. Methods: Quantum dot and colloidal gold probes have been assessed using anti-somatostatin labelling of secretory granules in human somatostatinoma tumour cells. Images of quantum dot labelled tissues were further examined under fluorescence light, confocal and super-resolution light microscopy. Results: Antibody conjugated quantum dot immunolabelling was highly specific and can be visualised by light and electron microscopy. Super-resolution microscopy displayed superior details of the target structures compare to fluorescence light and confocal microscopy. Conclusions: Quantum dot nanocrystals offer new possibilities for correlation studies incorporating light microscopy, electron microscopy and super resolution, providing a more sensitive and accurate assessment in diagnostic anatomical pathology.