Helen Kalirai

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.

Low molecular weight heat shock protein HSP27 is a prognostic indicator in rectal cancer but not colon cancer

Objective There are currently no biomarkers in routine clinical use for determining prognosis in rectal cancer. In a preliminary proteomic study, variation in the levels of heat shock protein 27 (HSP27) in colorectal cancer samples was observed. The expression of HSP27 in a cohort of 404 patients with colorectal cancer with a predominantly poor prognosis was characterised and an investigation was undertaken of whether the differences were related to clinical outcome. HSP27 levels in diagnostic rectal biopsies were compared with matched surgical samples to determine whether changes in expression occurred in the time between biopsy and surgery and to investigate whether preoperative radiotherapy affected expression. Finally, the relationship between HSP27 expression and outcome was examined in an independent cohort of 315 patients with a predominantly good prognosis. Methods HSP27 levels were determined using combined two-dimensional gel electrophoresis and tandem mass spectrometry (12 cases) and by immunohistochemistry using tissue microarrays of colorectal cancers sampled at surgery and 80 diagnostic rectal biopsies. Results HSP27 overexpression was strongly associated with poor cancer-specific survival in rectal cancer (n=205, p=0.0063) but not colon cancer (n=199, p=0.7385) in the cohort with a poor prognosis. Multivariate Cox regression confirmed nodal metastases (p=0.0001) and HSP27 expression (p=0.0233) as independent markers of survival in rectal cancer. HSP27 levels remained unchanged in the majority of cases (65/80, 81%) between diagnostic biopsies and matched surgical samples, regardless of whether patients had undergone preoperative radiotherapy. In the cohort with a good prognosis the association between HSP27 and survival was not observed in patients with either rectal (n=115; p=0.308) or colon cancer (n=200; p=0.713). Conclusion In a large cohort of patients with a poor prognosis, HSP27 is an independent marker of poor outcome in rectal cancer; its expression is not altered by neoadjuvant radiotherapy. This finding requires validation in an independent similar cohort of patients with rectal cancer. HSP27 levels merit evaluation as a stratification factor for treatment of rectal cancer.

Human breast epithelial stem cells and their regulation.

This review summarizes the current evidence for the existence of human breast stem cells and the pathways involved in their regulation, and discusses how the disruption of these pathways may result in the generation of a population of cells with the capacity for unlimited self‐renewal. Relevant data from mouse model systems are also discussed where appropriate. By understanding the molecular pathways that regulate self‐renewal of normal mammary stem cells, it may be possible to target the activation of these pathways in breast tumours. Copyright

Proteomic Analyses of the Vitreous Humour

The human vitreous humour (VH) is a transparent, highly hydrated gel, which occupies the posterior segment of the eye between the lens and the retina. Physiological and pathological conditions of the retina are reflected in the protein composition of the VH, which can be sampled as part of routine surgical procedures. Historically, many studies have investigated levels of individual proteins in VH from healthy and diseased eyes. In the last decade, proteomics analyses have been performed to characterise the proteome of the human VH and explore networks of functionally related proteins, providing insight into the aetiology of diabetic retinopathy and proliferative vitreoretinopathy. Recent proteomic studies on the VH from animal models of autoimmune uveitis have identified new signalling pathways associated to autoimmune triggers and intravitreal inflammation. This paper aims to guide biological scientists through the different proteomic techniques that have been used to analyse the VH and present future perspectives for the study of intravitreal inflammation using proteomic analyses.

Immunohistochemical assessment of mitotic count in uveal melanoma

Background/Aims:  The mitotic count of uveal melanomas correlates with the risk of metastatic death, but with haematoxylin and eosin (H&E)‐stained sections, it can be difficult to identify mitotic figures (MF) reliably. We investigated whether this measurement could be enhanced by immunohistochemistry, using the mitosis‐specific marker Phospho‐Histone H3 Ser10 (PHH3).

Single nucleotide polymorphism array analysis of uveal melanomas reveals that amplification of CNKSR3 is correlated with improved patient survival.

Metastatic death from uveal melanoma occurs almost exclusively with tumors showing monosomy of chromosome 3. However, approximately 5% of patients with a disomy 3 uveal melanoma develop metastases, and a further 5% of monosomy 3 uveal melanoma patients exhibit disease-free survival for >5 years. In the present study, whole-genome microarrays were used to interrogate four clinically well-defined subgroups of uveal melanoma: i) disomy 3 uveal melanoma with long-term survival; ii) metastasizing monosomy 3 uveal melanoma; iii) metastasizing disomy 3 uveal melanoma; and iv) monosomy 3 uveal melanoma with long-term survival. Cox regression and Kaplan-Meier survival analysis identified that amplification of the CNKSR3 gene (log-rank, P = 0.022) with an associated increase in its protein expression (log-rank, P = 0.011) correlated with longer patient survival. Although little is known about CNKSR3, the correlation of protein expression with increased survival suggests a biological function in uveal melanoma, possibly working to limit metastatic progression of monosomy 3 uveal melanoma cells.

Combined p53 and MDM2 biomarker analysis shows a unique pattern of expression associated with poor prognosis in patients with renal cell carcinoma undergoing radical nephrectomy.

Whats known on the subject? and What does the study add?

Uveal Melanoma Cell Lines Contain Stem-Like Cells That Self-Renew, Produce Differentiated Progeny, and Survive Chemotherapy

PURPOSE Uveal melanoma (UM) cells in high-metastatic risk tumors have an undifferentiated molecular signature indicative of a more primitive cellular phenotype. Given mounting evidence for the existence of cancer stem cells (CSC), the authors investigated whether UM cell lines retain a population of self-renewing tumorigenic cells. METHODS Single-cell cloning and spheroid culture studies were used to study the presence of a CSC-like population in two cell lines derived from the primary tumor (Mel270) and metastatic liver lesion (Omm2.5) of the same patient. RESULTS Mel270 and Omm2.5 cells exhibited distinct clonal morphologies in adherent culture akin to holoclones, meroclones, and paraclones. Holoclones were large colonies of tightly packed small cells, which could be serially passaged (> 10 generations) to produce colonies of all three types; paraclones were small colonies of flattened cells that could be passaged for only one or two generations to produce further paraclones. Mel270 and Omm2.5 cells surviving cisplatin treatment produced significantly more holoclones than untreated cells (P < 0.05), suggesting enrichment for this CSC-like subpopulation. Mel270 and Omm2.5 cells also formed melanomaspheres (MS) when grown at clonal density in nonadherent culture. MS possessed self-renewal capacity to generate further MS and when replated to adherent culture yielded colonies of all three types. Mel270 and Omm2.5 holoclones and MS also demonstrated antigenic heterogeneity expressing markers associated with both a primitive migratory neural crest phenotype, and a more differentiated phenotype. CONCLUSIONS These data suggest the presence in UM cell lines, of a CSC-like subpopulation with enhanced self-renewal and proliferative capabilities that could more appropriately model therapeutic efficacy.

Proteomics of Uveal Melanomas Suggests HSP-27 as a Possible Surrogate Marker of Chromosome 3 Loss

PURPOSE To compare the proteomic profiles of primary uveal melanomas, with and without loss of chromosome 3. METHODS Frozen specimens from three uveal melanomas with disomy 3 and from four tumors with monosomy 3, according to fluorescence in situ hybridization (FISH) analysis, were subjected to high-resolution, two-dimensional (2-D) gel electrophoresis. The protein expression profiles of the two uveal melanoma cytogenetic groups were compared: Proteins that differed significantly were excised and analyzed by tandem mass spectrometry. Differentially expressed proteins were further analyzed with Western blot analysis. An independent cohort of 41 formalin-fixed, paraffin-embedded (FFPE) uveal melanomas, whose chromosome 3 status had been determined by multiplex ligation-dependent probe amplification (MLPA), was examined for the appropriate antigens by immunohistochemistry. RESULTS Four protein spots were 1.5-fold (Students t-test, P < 0.05) differentially expressed in the two uveal melanoma types: two spots were overexpressed in the disomy 3 group compared with the monosomy 3 group, whereas two spots were underexpressed. Identification of the four spots yielded nine proteins. Western blot analysis confirmed the results for heat shock protein (HSP)-27, vimentin, and pyruvate dehydrogenase beta (PDHB), with a statistical significance for the first two proteins. HSP-27 was significantly downregulated, whereas vimentin was upregulated in the monosomy 3 tumors (Students t-test, P = 0.003 and P = 0.005, respectively). Immunohistochemistry confirmed low-to-negative HSP-27 protein expression in monosomy 3 uveal melanomas (Students t-test; P = 0.011). CONCLUSIONS Low-to-negative HSP-27 protein expression in uveal melanoma correlates strongly with monosomy 3. Further validation is necessary to determine whether immunohistochemical assessment of HSP-27 expression correlates with metastatic mortality.

SERPINE1 and SMA expression at the invasive front predict extracapsular spread and survival in oral squamous cell carcinoma.

Background:Extracapsular spread (ECS) in cervical lymph nodes is the single-most prognostic clinical variable in oral squamous cell carcinoma (OSCC), but diagnosis is possible only after histopathological examination. A promising biomarker in the primary tumour, alpha smooth muscle actin (SMA) has been shown to be highly prognostic, however, validated biomarkers to predict ECS prior to primary treatment are not yet available.Methods:In 102 OSCC cases, conventional imaging was compared with pTNM staging. SERPINE1, identified from expression microarray of primary tumours as a potential biomarker for ECS, was validated through mRNA expression, and by immunohistochemistry (IHC) on a tissue microarray from the same cohort. Similarly, expression of SMA was also compared with its association with ECS and survival. Expression was analysed separately in the tumour centre and advancing front; and prognostic capability determined using Kaplan–Meier survival analysis.Results:Immunohistochemistry indicated that both SERPINE1 and SMA expression at the tumour-advancing front were significantly associated with ECS (P<0.001). ECS was associated with expression of either or both proteins in all cases. SMA+/SERPINE1+ expression in combination was highly significantly associated with poor survival (P<0.001). MRI showed poor sensitivity for detection of nodal metastasis (56%) and ECS (7%). Both separately, and in combination, SERPINE1 and SMA were superior to MRI for the detection of ECS (sensitivity: SERPINE1: 95%; SMA: 82%; combination: 81%).Conclusion:A combination of SMA and SERPINE1 IHC offer potential as prognostic biomarkers in OSCC. Our findings suggest that biomarkers at the invasive front are likely to be necessary in prediction of ECS or in therapeutic stratification.