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

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Presentation on theme: "Specialist Biomedical Scientist Cellular Pathology Phospho-histone H3 staining of uveal and choroidal melanomas."— Presentation transcript:

1 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


6 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)

7 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

8 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

9 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

10 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

11 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.

12 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

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

14 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

15 Materials and Methods: Counting Mitoses 30 high power fields (x40 lens) Corresponds to an area of 10mm 2 Numbers obtained from H & E staining and PHH3 staining were compared Counting carried out by Ophthalmic Histopathologist

16 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

17 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

18 Results: Clinical and Histological Parameters










28 Still alive 20 years laterDied 18 months after enucleation

29 Results: Mitotic Figure Staining




33 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

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

35 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) Results: Survival Time

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

37 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)

38 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

39 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

40 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 Discussion

41 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

42 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)

43 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


45 Questions?

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