1 / 45

Joy Elizabeth Martindale

Joy Elizabeth Martindale. Specialist Biomedical Scientist Cellular Pathology. Phospho-histone H3 staining of uveal and choroidal melanomas. Anatomy of the eye. As normal as it gets. Iris. Ciliary Body. Choroid. Anatomy of the eye. Risk Factors.

camden
Download Presentation

Joy Elizabeth Martindale

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Joy Elizabeth Martindale Specialist Biomedical Scientist Cellular Pathology Phospho-histone H3 staining of uveal and choroidal melanomas

  2. Anatomy of the eye

  3. As normal as it gets Iris Ciliary Body Choroid

  4. Anatomy of the eye

  5. Risk Factors • Similar risk factors for cutaneous melanomas • Incidence of uveal melanomas is highest in the white population with between 4 to 10 cases per million • UV light may be a risk factor • Congenital ocular melanocytosis • Familial atypical mole and melanoma syndrome (FAMM)

  6. Prognostic Factors: Clinical • Age and Sex • Rare in childhood • Risk increases with age: most melanomas occur in patients of late middle age • Slight male predominance, but this is not statistically significantly linked to survival • Tumour location • Location of the anterior margin of the tumour is an important predictor of prognosis and survival • Ciliary body tumours carry worst prognosis • Iris tumours have lowest mortality rate • Tumours adjacent to the optic disc have worse prognosis • Tumour size • Larger tumour size indicates worse prognosis • Smaller tumours capable of causing death through metastasis

  7. Prognostic: Cytogenetic and Molecular • Chromosome aberrations: • Monosomy 3 is predictor of poor prognosis • Amplification of 8q is associated with reduced survival • Loss of 1p is associated with increase in mortality from metastases • Gain of 6p is associated with good prognosis • Loss of tumour suppressor genes • P53 • Rb • Mutations in tumour promoter genes correlate with prognosis • Poor prognosis: • DDEF1 • NBS1 • Better prognosis • C-myc

  8. Prognostic Factors: Histopathological • Cell type • Consistent prognostic factor • Spindle cell; epithelioid cell; mixed cell • Spindle = best prognosis • Epithelioid = worst prognosis • Microvascular patterns and microvascular density (MVD) • Microvascular patterns: loops found in 60% of melanomas and associated with poorer prognosis • High MVD = associated with shortened survival • Cell matrix interactions • Expression of MMP-2 decreases survival • EGFR and IGF-1R linked with metastases • Number of mitotic figures • Correlates with mortality

  9. PHH3 is a Mitosis-specific Marker • Core protein Histone H3 • Involved in maintaining the integrity of the DNA double helix within senescent cells • Is phosphorylated at the serine 10 residue during mitosis • Phosphorylation only occurs during mitosis and not during apoptosis • Antibody which detects this phosphorylation event has been developed and used to detect mitotic figures in various different tumours • Astrocytomas • Meningiomas • Uveal and choroidal melanomas

  10. Aims • To perform immunohistochemical (IHC) staining for phospho-histone H3 (PHH3) in choroidal melanoma • To compare staining of mitotic figures using H & E staining and IHC staining with the PHH3 antibody • To see if IHC staining correlates with clinical outcome, histopathological features and presence or absence of monosomy 3.

  11. Materials and Methods: Population • Previously diagnosed uveal melanomas (1973 – 1992) • 60 enucleations • 50 local resections • 1 exenteration • Groups • 1 = Metastasising • 2 = Non-metastasising • Tissues fixed in glutaraldehyde or formalin and embedded in paraffin wax

  12. Materials and Methods: H&E Staining • Sections cut at 3µm • Harris’ haematoxylin • Alcoholic eosin • Automated staining

  13. Materials and Methods: IHC Staining • Bond™Max Automated Immunohistochemistry Vision Biosystem • IHC Protocol J • Bond dewax solution • Epitope Retrieval solution 2 • Primary antibody: PHH3 • Post primary alkaline phosphatase: rabbit anti-mouse IgG • Polymer alkaline phosphatase: anti-rabbit IgG • Fast red chromogen • Haematoxylin counterstain

  14. Materials and Methods: Counting Mitoses • 30 high power fields (x40 lens) • Corresponds to an area of 10mm2 • Numbers obtained from H & E staining and PHH3 staining were compared • Counting carried out by Ophthalmic Histopathologist

  15. Materials and Methods: Statistical Analyses • Two staining techniques compared using Wilcoxon Signed Rank Test • Level of significance set at p < 0.05 • Survival time for patients with metastasising melanomas compared using Wilcoxon Rank Sum Test and the Kaplan- Meier non-parametric distribution analysis • Level of significance set at p < 0.05 • Correlations between the number of mitotic figures (using PHH3) and histological parameters examined using the Kruskall-Wallis non-parametric test • All completed using Minitab software

  16. Results: • 40 cases of metastasising melanomas • Mean age 56.2 years (range 16.92 – 79.62) • 20 cases of non-metastasising melanomas • Mean age of 52.8 (range 18 – 78) • Ages of patients were statistically similar • Male to female distribution was similar • No significant differences in treatments

  17. Results: Clinical and Histological Parameters

  18. Results: Clinical and Histological Parameters

  19. Still alive 20 years later Died 18 months after enucleation

  20. Results: Mitotic Figure Staining

  21. Results: Mitotic Figure Staining

  22. Results: Mitotic Figure Staining

  23. Results: Mitotic Figure Staining

  24. Results: Assessment of Mitotic Rate • Comparison of staining with H&E and PHH3 • Non-metastasising melanoma : p = 0.009 • Metastasising melanoma: p < 0.0001 • All samples: p < 0.0001

  25. Results: Prediction of Survival Time • Statistically significant differences between the two staining methods: therefore for subsequent analyses, data obtained from PHH3 were used

  26. Results: Survival Time • Number of mitotic figures were grouped: • < 5 • 5 – 10 • > 10 • Groupings of mitotic figure counts were compared using non-parametric tests: • Wilcoxon Rank Sum Test • Kaplan-Meier • No statistically significant differences between survival times of patients in each grouping (p = 0.116)

  27. Results: Kaplan-Meier • Indication that patients with >10 mitoses survive for a shorter length of time

  28. Prediction of Mitoses from Histological Features • Kruskal-Wallis non-parametric test determined correlations between mitoses and other features • Balloon cells (p = 0.036) • Pigment (p = 0.584) • Number of lymphocytes (p = 0.268) • Cell type (p = 0.986) • Presence of vascular loops (p = 0.534) • Necrosis (p = 0.832) • Invasion of tumour into surrounding tissue (p = 0.820) • Largest tumour dimension (p = 0.159) • Survival time (p = 0.187) • Monosomy 3 (p = 0.199)

  29. Discussion • PHH3 staining is a valid technique for demonstrating mitotic figures • The number of mitotic cells in some tumour types is a good indicator of metastatic potential

  30. Discussion • Using this technique: • Mitotic counts were elevated in 17 out of 40 (42.5%) cases of metastasising uveal melanomas when compared with H&E staining • Mitotic counts were elevated in 10 out of 20 (50%) of non-metastasising uveal melanomas when compared with H&E staining • Significant differences in the number of mitotic figures when stained with PHH3 and H&E • Non-metastasising melanomas: p = 0.0009 • Metastasising melanomas: p < 0.0001 • Most likely reason: there are significantly more mitoses in metastasising melanoma than in non-metastasising melanoma

  31. Discussion • 25 cases failed to stain with PHH3 • Historic tissues were used (some up to 39 years old) • Tissues lose antigenicity over time with long term storage • Diaminobenzidine (DAB) chromogen staining is masked by the melanin pigment in the tumour • Double-staining will overcome this, but it is time-consuming • In this project, the red chromogen was used to distinguish between melanin pigment and mitotic figure staining

  32. Discussion • IHC staining: • Allows easier recognition of mitotic figures (when compared to H&E staining) • Reduces the time required for counting mitotic figures by as much as 50.4% • Is relatively cheap to carry out (although the costs of individual reagents is more expensive than H&E staining) • Is less labour intensive than H&E staining (automated staining) • Facilitates counting of mitotic figures by BMS staff, allowing the histopathologist to undertake other duties

  33. Discussion • Correlation between number of mitoses and: • Presence of balloon cells (p=0.036) • No correlation between number of mitoses and: • Pigment (p = 0.584) • Number of lymphocytes (p = 0.268) • Cell type (p = 0.986) • Presence of vascular loops (p = 0.534) • Necrosis (p = 0.832) • Invasion of tumour into surrounding tissues (p = 0.820) • Survival times (p = 0.187) • Presence or absence of monosomy 3 (p=0.199)

  34. Future Work • Using PHH3 antibody to demonstrate the number of mitoses in other tumour types • GIST • Developing an international standardised method for counting mitoses may be useful in order to aid classification of tumours • Comparison of the times taken for Histopathologist and BMS to count mitotic figures

  35. Questions?

More Related