Br J Ophthalmol 97:40-46 doi:10.1136/bjophthalmol-2012-302261
  • Clinical science

Fluorescence in situ hybridisation (FISH) in histologically challenging conjunctival melanocytic lesions

  1. Ian G Rennie3
  1. 1National Specialist Ophthalmic Pathology Service, Department of Histopathology, Royal Hallamshire Hospital, Sheffield, UK
  2. 2Sheffield Diagnostic Genetics Service, Sheffield Children's NHS Foundation Trust, Sheffield, UK
  3. 3Academic Unit of Ophthalmology and Orthoptics, University of Sheffield, Sheffield, UK
  1. Correspondence to Dr Hardeep Singh Mudhar, National Specialist Ophthalmic Pathology Service, Department of Histopathology, Royal Hallamshire Hospital, Glossop Road, Sheffield S10 2JF, UK; hardeep.mudhar{at}
  • Received 27 June 2012
  • Revised 15 August 2012
  • Accepted 8 October 2012
  • Published Online First 8 November 2012


Background Even in experienced hands, the classification of some melanocytic lesions of the conjunctiva remains challenging. In skin pathology, the recent application of fluorescence in situ hybridisation (FISH) has been demonstrated to be of use for the analysis and diagnosis of ambiguous melanocytic neoplasms of the skin. This study set out to evaluate this method on seven prospective conjunctival cases that were histologically equivocal.

Methods 18 unequivocal retrospective melanocytic controls were exposed to FISH. Commercially available probes assessing copy numbers of RREB1 (6p25), MYB (6q23) and CCND1 (11q13) genes compared with CEP6 (a chromosome six centromeric reference point) were used. After control verification, seven prospective, equivocal cases were identified and exposed to FISH.

Results There was complete correlation between FISH result and the control section histopathology report. Control cases of melanoma cases were all positive for FISH and control benign lesions were negative. Of the seven equivocal cases, five were positive and classed as invasive melanoma or melanoma-in situ, one was negative and one tetraploid, classed as negative (these last two cases were classed as naevi with careful clinical observation).

Conclusions FISH is very useful in classifying equivocal conjunctival melanocytic lesions, especially those with atypical junctional activity and naevoid melanocytic proliferations of the conjunctiva.


Melanocytic lesions of the conjunctiva, comprising benign naevi and melanomas (in situ and invasive), constitute a substantial proportion of the specialist ophthalmic histopathology workload, especially if allied to a clinical ocular oncology ophthalmology service. While most conjunctival melanocytic lesions are straightforward to diagnose morphologically, there remain a small proportion of cases that are difficult to categorise from morphology alone. These cases fall into the following categories.

  1. The presence of linear and nested junctional melanocytic proliferation over a naevus, in which the issue is whether the junctional activity is permissible according to ‘site specific atypia’ or indicates malignant potential.

  2. ‘Naevoid’ atypical stromal melanocytic lesions where melanoma requires exclusion.

  3. The significance of linear melanocytes along the basal layer of the conjunctiva after treatment.

It is important to derive an accurate diagnosis given the difference in prognosis and treatment between a conjunctival benign naevus and conjunctival in situ/invasive melanoma. A benign naevus diagnosis leads invariably to discharge from the eye clinic whereas a malignant diagnosis requires further treatment (eg radiation therapy, topical chemotherapy) and follow-up for life.

Recently, fluorescence in situ hybridisation (FISH) has been demonstrated to be of use for the analysis and diagnosis of ambiguous melanocytic neoplasms of the skin.1 ,2 Commercially available probes assessing copy numbers of RREB1 (6p25), MYB (6q23) and CCND1 (11q13) genes, compared with CEP6 (a chromosome 6 centromeric reference point), have shown good sensitivity and specificity for cases that displayed equivocal histological features.2

A recent study validated FISH technique for conjunctival melanocytic lesions and was useful in establishing a diagnosis of melanoma in two conjunctival biopsies where the histology was problematic due to fragmentation, tangential cutting and confounding inflammation within the tissues.3

In this report, we describe our experience with seven cases of conjunctival melanocytic lesions where FISH proved to be of utility when considered alongside conventional histology in reaching a firmer diagnostic conclusion.

Materials and methods

The project was approved and registered as a service evaluation, number 984 by the Clinical Governance Department at Sheffield Children's NHS Foundation Trust. Control tissue was retrieved from the Royal Hallamshire Hospital Histopathology paraffin block archive. Control tissue comprised unequivocal examples of conjunctival benign naevi, benign intraepithelial melanocytic proliferations (melanosis without atypia) and unequivocal examples of in situ and invasive melanoma from 18 samples. Within this study, seven test cases were identified prospectively and were judged to show equivocal histological features.

FISH was performed according to the following protocol: Paraffin wax embedded sections (4 µm thick) mounted on X-tra slides (Leica Microsystems, UK) were dewaxed for 2×10 min, dehydrated for 5 min in 50 ml ethanol and then washed in 50 ml of 0.2 M HCl. The slides transferred to 50 ml Zymed heat pretreatment solution (Zymed laboratories Inc, South San Francisco, California, USA) at 95°C for 80 to 110 min. A quantity of 90 µl of Zymed digestion enzyme solution (Zymed laboratories Inc, South San Francisco, California, USA) was then applied to a 22×50 mm coverslip and overlaid on to the slide. The slide was then incubated at 38°C in a wet box for 50–70 min. Slides were then washed before being dehydrated through an ethanol series. The slides were then left to dry before probe application. The sample DNA and probe DNA were co-denatured at 72°C for 5 min and then allowed to hybridise at 37°C overnight on a PTC-200 thermal cycler (MJ Research, Waltham, Massachusetts, USA). Slides were then washed; dehydration was performed, as before, and the slides were air dried in darkness. The slides were coverslipped with 20 µl of counterstain (20 µl mounting medium with 4',6-diamidino-2-phenylindole (DAPI) in 1000 µl mounting medium for fluorescence; Vector Laboratories, Burlingame, California, USA). FISH probes used consisted of four loci: RREB1 at 6q23, 6 centromere, MYB at 6p25 and CCND1 at 11q13; these were incorporated into a melanoma panel of probes supplied by the Abbott Vysis Corporation (Downers Grove, Illinois, USA). Then, 4 µm thick paraffin wax sections from histopathology archive blocks were received, fixed in 10% buffered formalin. The processing of the paraffin wax sections for FISH was as previously reported (Ellis et al 2005).4 Scoring was performed double-blinded. Examination of 30 nuclei from at least three different areas of each section was performed by each of two independent analysts. Appropriate areas were identified at ×400 magnification and scored at ×1000 by the first analyst using an Olympus BX51 fluorescence microscope (Olympus UK, Essex, UK). Recorded areas were relocated and scored by the second analyst. The expected signal pattern for the probe set was 2:2:2:2. The score of probe signals was lower than the theoretical score because of nuclear truncation caused by sectioning at 4 μ and by colocalisation of signals, as an artefact of the technique. Representative multiplane Z-stack images of all successful hybridisations were captured at ×1000 magnification using the Genetix Cytovision software (Genetix Cytovision, Leica Microsystems, New Milton, UK) and an Olympus BX61 semiautomated fluorescence microscope. The appropriate areas for scoring were selected by the histopathologist, and the areas indicated on H&E or Imelda stained slides were relocated on FISH section. Where clearly in situ and invasive areas of tumour were available, 10 nuclei from three separate areas of tumour (30 in total) were scored. Where individual abnormal melanocytes were indicated, these areas were relocated on FISH section; individual nuclei with abnormal FISH pattern were selected and scored individually. The average score for these selected cells was then used to calculate FISH classification of positive or negative. Classification of negative or positive FISH score was calculated according to the criteria given by Gerami et al 2009.2 Briefly, a FISH result was positive if any of the following criteria were met: gain of 6p25 (RREB1) relative to CEP6 greater than 55%, gain of 6p25 (RREB1) greater than 29%, loss of 6p23 (MYB) relative to CEP6 greater than 40% or gain of 11q13 (CCND1) greater than 38%.2


The clinical details, histology and FISH findings for the 18 control samples are listed in table 1. There were 13 female subjects (age range 14–88 years) and five male subjects (age range 15–78 years).

Table 1

FISH technique verification data on the 18 retrospective control cases

Once FISH technique had been verified on the retrospective control tissue, the technique was applied to seven prospective test cases with equivocal histology. These comprised four male subjects (age range 22–46 years) and three female subjects (age range 47–69 years). Six out of seven presented with bulbar conjunctival melanocytic lesions and one in the plica region. The commonest clinical reason for presentation was a change in size of the lesion. Five of the seven index cases were FISH positive. One was negative and one was tetraploid and considered to be negative. Results are detailed in table 2.

Table 2

Data of seven prospective cases tested with FISH


This study has corroborated the findings of Busam et al3 that FISH assay can be used on conjunctival melanocytic lesions and that conjunctival melanomas have similar genetic aberrations to skin melanomas (unlike uveal melanomas that have their distinct chromosomal changes, principally of chromosomes 3 and 8). We have further expanded the role of FISH by applying it to diagnostically challenging/ambiguous conjunctival melanocytic lesions. Conjunctival melanocytic lesions can be challenging even to experienced histopathologists; they can display certain atypical features, such as asymmetry at low power, confluent nests of melanocytes and melanocyte discohesion, which if partially developed makes it difficult to classify the behaviour of the lesion.5–7

In case 1, the stromal nodule was monotonous but composed of cells with low nuclear to cytoplasmic ratio and inconspicuous nucleoli. However, maturation was lacking and there was an adjacent lymphatic channel containing similar melanocytic cells (figure 1A–C). The differential diagnosis lay between an atypical naevus and a melanoma. Positive FISH findings on the main stromal nodule (figure 1D) and in the intralymphatic cells (not shown) supported a diagnosis of melanoma of the conjunctiva with lymphatic permeation.

Figure 1

(A and B) H&E—Patient 1: Showing the stromal nodule, with (B) at higher power. Bland monotonous cells with low N to C ratio, inconspicuous nucleoli and bland nuclei. (C) Patient 1: The arrow points to a lymphatic channel stained immunohistochemically with lymphatic endothelial cell marker D240 (brown), which encloses melanoma cells. (D) Patient 1: Fluorescence in situ hybridisation (FISH) gain of RREB1 (red signal) in the stromal melanocytes. (E and F) H&E—Patient 2: Showing an atypical/confluent junctional melanocytic proliferation ((F) shows higher power of junctional proliferation) over an otherwise benign naevus, part of which is shown in (G). (H) Patient 2: FISH with gain of RREB1 (red signal) in abnormal melanocytes. (I) Patient 2: FISH with gain of CCND1 (green signal) in upper cell. Lower cell is normal with 2 copies of each chromosome. This gain was seen in scattered cells along the conjunctival epithelium on rebiopsy. (J and K) H&E—Patient 3: Showing the abnormal junctional component in (J) and the benign naevus component deeper in the stroma in K. (L) Patient 3: FISH showing gain in CCND1 (green signal) and RREB1 (red signal). (M) Patient 3: Immunohistochemistry of Melan A showing a scattered basal population of melanocytes after topical chemotherapy (mitomycin C). These cells show gain of CCND1 (green signal) and RREB1 (red signal) illustrated in (N). (O) H&E—Patient 4: Showing junctional atypia. (P) H&E—Patient 4: Showing the banal stromal naevus component. (Q) Patient 4: FISH of the junctional component shows a normal pattern with 2 copies of each signal. (R) H&E—Patient 5: Showing rounded nests of melanocytes beyond the cystic stromal naevus component shown in (S). (T) Patient 5: FISH of the junctional component shows a tetraploidy (4 signals per colour). (U) H&E—Patient 6: First biopsy taken and thought to represent a benign naevus. On retrospective examination, this architecturally atypical trabecular stromal component was present. (V) Patient 6: FISH analysis on the section (U) shows loss of MYB (one yellow signal only). (W) Patient 6: Recurrence of lesion 7 years later showing similar distribution of melanocytes in stroma compared with (V). (X) Patient 6: FISH analysis on the recurrence shows loss of MYB (one yellow signal only), identical to (V).

Figure 1


Cases 2 and 3 displayed confluent nested and linear, cytologically atypical melanocytic proliferations at the epithelial–stromal junction, which were difficult to place neatly into ‘site specific’ permissible atypia. These cases proceeded to FISH which showed that the junctional areas were FISH positive (figure 1E–L) compared with the naevoid areas that were FISH negative (the latter providing a useful internal comparator), supportive of a diagnosis of in situ melanoma evolving from a conjunctival naevus. In case 2, a subsequent biopsy showed junctional melanocytes of a similar nature to those in the index biopsy. While FISH was statistically interpreted as negative according to the Gerami et al criteria,2 the presence of scattered melanocytes, exhibiting chromosomal copy number aberrations, was noted and interpreted as residual in situ melanoma (figure 1I, upper cell). In case 3, residual intraepithelial melanocytes, in a second biopsy after a course of topical chemotherapy (mitomycin C), were FISH positive (figure 1M,N).

Cases 4 and 5 exhibited occasional nests beyond the main naevus component. The nests in case 4 were negative for FISH (figure 1Q), and in case 5 tetraploidy was identified (figure 1T). In both cases, the naevi were reported as architecturally aberrant but with no evidence of malignancy, with advice to careful follow-up. Tetraploidy has been detected in skin naevi on FISH testing and is often a source of false positivity in FISH analysis of melanocytic lesions.8 ,9 The presence of tetraploid cells is readily identifiable as the increased signal pattern affects all probes in FISH panel. However, in order to avoid this pitfall in FISH analysis of these cases, FISH assay and interpretation was performed by experienced FISH analysers with this phenomenon in mind.

Cases 6 and 7 illustrate the histopathological interpretation issues with atypical naevoid stromal components. Case 6 (figure 1U–X) was diagnosed as a ‘compound naevus’ previously which recurred at the same location on the bulbar conjunctiva 7 years later. The latter lesion showed FISH positivity in the nevoid melanocytes, arranged as trabeculae in the stroma (figure 1X). Retrospective examination of the first biopsy that had been designated as a compound naevus 7 years previously showed a trabeculated arrangement of melanocytes in the stroma that were also FISH positive (figure 1V), supportive of an interpretation of recurrent invasive melanoma. Post-treatment biopsies of this case were analysed using FISH and, although were considered statistically FISH negative, revealed some individual intraepithelial melanocytes with an aberrant FISH signal related to the abnormal FISH pattern detected in the pretreatment in situ component. The clinicians were advised that this population could act as a potential nidus for recurrent disease, meriting regular close review (figure not shown).

The index biopsy in case 7 showed a longstanding naevus, with an atypical stromal and junctional component which were both FISH positive (figure 2A–C). On wider excision, the case was reported as residual in situ melanoma with residual benign stromal naevus. When FISH was applied to the re-excision specimen, surprisingly, the stromal naevoid component was positive for FISH (figure 2D, E), along with the residual in situ component. Review of the H&E revealed that this morphological undercall had resulted from not appreciating subtle changes in nuclear and nucleolar features.

Figure 2

(A) H&E—Patient 7: Atypical junctional component. (B) H&E—Patient 7: Scattered atypical stromal melanocytes (arrowed). (C) Patient 7: Fluorescence in situ hybridisation (FISH) shows a gain in RREB1 (red signal) and CCND1 (green signal) in junctional component. An identical signal was present in the scattered atypical stromal melanocytes. (D) Patient 7: Re-excision specimen showing a naevus stromal component, adjacent to cysts. (E) Patient 7: FISH on section described in D showing gains in RREB1 (red signal) and CCND1 (green signal).

In our centre, we do not test every conjunctival melanocytic lesion with FISH. We only reserve FISH testing for selected cases with equivocal histology after thorough examination of the clinical history and a detailed scrutiny of the H&E stained slides. Most cases, in our experience, can be diagnosed as benign naevi or malignant melanoma on the basis of morphology alone. We still rely heavily on morphology and believe that FISH cannot replace this basic modality. We have adopted the approach, outlined in a recent review: ‘if the histopathological diagnosis may very well be melanoma, a positive FISH result is supportive. A negative FISH result should not be taken as dismissal’.10

In summary, this FISH study has been extremely useful for highlighting and diagnosing ambiguous melanocytic lesions that fall into the naevoid category, those with ‘uncomfortable’ junctional activity thought to be beyond site specific atypia and in establishing the nature of basal melanocytes in re-excisions or postchemotherapy samples. We have not yet examined the prognostic significance of these FISH positive cases, especially in light of recent cases from the skin literature that FISH positivity confers a worse prognosis11 nor have we examined any large cohorts of postchemotherapy associated biopsies to ascertain the biology of those residual melanocytes that persist. These points will be addressed in future studies.


  • Contributors HSM is the main author who conceived the study and wrote the paper. KS, PT, AW and NA performed FISH analysis and data collection/interpretation and suggested changes to the paper. IR contributed to the intellectual content of the paper.

  • Funding Funding was provided by Sheffield Diagnostic Genetic Service, Sheffield Children's Hospital for the purposes of a Service Evaluation.

  • Competing interests None.

  • Ethics approval This service evaluation was approved and registered as number 984 by the Clinical Governance Department at Sheffield Children's NHS Foundation Trust on 31 March 2010.

  • Provenance and peer review Not commissioned; externally peer reviewed.