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British Journlal of Ophthalmology, 1980, 64, 453-456

Tumours of the anterior uvea. II. Intranuclearcytoplasmic inclusions in malignant melanomaof the irisM. S. NOOR SUNBA, AMJAD H. S. RAHI, AND GWYN MORGANFrom the Departmenit of Pathology, Institiute of Ophthalmology, London

SUMMARY Intranuclear cytoplasmic inclusions have been described previously in both human andanimal tissues. So far as we are aware they are described in detail for the first time in this paper in60 cases of malignant melanoma of the iris, usually in spindle cells. The light microscopic appear-

ances are described, and electron microscopic studies of selected cases show clearly the genesis ofthese inclusions by cytoplasmic invagination of the nuclear membrane. Their significance is dis-cussed. It is of interest that we have seen identical inclusions in malignant melanomas of the ciliarybody, choroid, and conjunctiva but have not as yet reported our findings.

Although nuclear inclusions have been describedon a number of occasions,1-4 both in human andanimal tissues, when they were assumed to be ofviral origin, it was not until the introduction of theelectron microscope that the largest group ofnuclear inclusions were found to be cytoplasmicinvaginations of the nuclear membrane.5 In anelectron microscopic study of a case of tapiocamelanoma of the iris6 curious intranuclear pigmentinclusions were described. A brief reference hasalso been made to cytoplasmic invaginations of thenucleus in a case of iris melanoma,7 but the authorsdid not fully document the cytoplasmic inclusionsthat we are describing in the present paper, thepurpose of which is to report in detail the incidenceand morphology of these nuclear inclusions inmalignant melanomas of the iris by both light andelectron microscopy and to trace their genesis bynuclear invagination.

Materials and methods

During the light microscopic study of routinesections of 199 cases of malignant melanoma ofthe iris, stained by haematoxylin and eosin, nuclearinclusions were observed in 60 cases. Some of thelatter were examined after staining by the periodicacid Schiff (PAS) method before and after diastasetreatment, by methyl green-pyronin, by the Fontanastain, and with amino acid stains. In addition 1 ,umthick plastic sections of some cases were stained with

Correspondence to Dr A. H. S. Rahi, Institute of Ophthal-mology, Judd Street London WC1H 9QS.

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W..Fig. 1 Malignant melanoma of iris, showing intranuclearinclusions of varying size and shape. One nucleus (arrow)contains 2 inclusions. (Haematoxylin-eosin, x 245.)

toluidine blue, and paraffin embedded sections ofother melanomas were postfixed in osmium tetroxidefor electron microscopic examination by the JEOLJEM-IOOC instrument. One malignant melanoma ofthe choroid was freshly fixed in glutaraldehyde andblocked in Araldite for similar studies.

Results

As stated in the previous paragraph, intranuclearinclusions were seen on light microscopy in 60 cases,

40 being females and 20 males. Fifty-seven of thetumours were spindle cell melanomas and 3 were

of the mixed type. Inclusions were usually found inspindle cells. Some were small and others almostfilled the nucleus; they varied in shape betweenspindle, spherical, and bizarre, and some nucleicontained two inclusions (Fig. 1). Some were situated

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Fig. 2 Plastic section, showing centrally placedinclusion (arrow). (Toluidine blue, x 1440.)

centrally in the nucleus (Fig. 2), and others werefound at one pole, which was often distended. Notinfrequently both the nucleus and cytoplasm becomedistended. The inclusions had a limiting membranesimilar to the nuclear membrane and sometimescontained fine granules resembling melanin granules.

Inclusions stained with eosin with a varyingdegree of intensity and were PAS-positive both

before and after diastase treatment. With the methylgreen-pyronin stain both the inclusions and thecytoplasm of the cells stained red. With the Fontanastain a few inclusions contained faintly stainingmelanin granules. The inclusions stained positivelywith varying intensity for tyrosine and for thesulphur-containing amino acids (Table 1).8

Plastic sections and electron microscopic sectionsshowed clearly that the inclusions were of cytoplas-mic origin and followed invagination of the nuclearmembrane (Fig. 3). They contained cytoplasmicstructures such as mitochondria, premelanosomes,

Table I Histochemical staining of intranuclear inclusions

Method (Pearse)) Result

1 PAS Positive2 Diastase-PAS Positive3 Fontana Positive4 Tyrosine (DC) Positive5 Sulphur-containing Positive

amino acids: cystine,cystein, methionine

6 Tryptophan (DMAB) Negative7 Arginine (Sakaguchi) Negative8 Methyl green-pyronin Pyroninophilic

*- ,,< ,

Fig. 3 Electron micrograph, showing the genesis of the inclusions by nuclear invagination ( x 8750). Inset areplastic sections showing similar features. (Toluidine blue, x 1600.)

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M. S. Noor Sunba, Amjad H. S. Rahi, and Gwyn Morgan

melanosomes, microfilaments, ribosomes, roughendoplasmic reticulum, and annulate lamellae (Fig.4), and were separated from the nuclear matrix bya double-layered membrane, the outer aspect ofwhich consisted of condensed chromatin similarto that of the inner aspect of the nucleus. The innerafpect of the membrane was studded with ribosomesas is the outer aspect of the nuclear envelope.Eiectron microscopy of a malignant melanoma ofthe choroid showed well preserved mitochondria,premelanosomes, and melanosomes (Fig. 5).

Discussion

Although cytoplasmic intranuclear inclusions similarto those described in this paper have been reportedpreviously,7 9-13 to our knowledge this is the firstoccasion that they have been described in detail inmalignant melanomas of the iris. We should like toadd that we have also seen very similar inclusionsin malignant melanomas of the ciliary body, choroid,and conjunctiva.The intranuclear inclusions found in malignant

melanomas of the iris varied in size, shape, site, anddensity of staining. Although we did not carry outhistochemical studies for enzymes, which arefound exclusively in the cytoplasm, electron micro-scopy showed quite clearly that they were cyto-plasmic invaginations of the nuclear membrane, sothat in effect they are pseudoinclusions rather thantrue inclusions.The genesis of the nuclear invagination is un-

known. Some authors14 found the inclusions in largecells which contained much cytoplasm. Leduc andWilson5 consider that the invagination develops asan attempt to maintain the normal ratio of nuclearsurface to nuclear volume, while Sobel et al.15consider that the majority of inclusions can beexplained by the swollen cytoplasm extruding intothe nucleus. Serber'0 studied the development ofcytoplasmic intranuclear inclusions in the pituitarygland of experimental animals and found that theywere related to the process of aging and probablydeveloped due to a deficiency of sex hormones.Although we do not understand the genesis of

these intranuclear cytoplasmic inclusions, we thinkthat there may be an increase in the effective nuclearsurface and the associated cytoplasm which leadsto the invagination of the nuclear membrane by thecytoplasm. The significance of the annulate lamellaein some inclusions is not clear, but similar structuresin the cytoplasm of normal epithelium have beenidentified with active protein synthesis."' It istempting to postulate, therefore, that these struc-

tures play a similar role in tumour cells. An activeprotein synthesis with consequent increase incytoplasmic volume could lead to the cytoplasmicinvagination of the nuclear membrane. Because ofthe finding of inclusions of different shape and sizein sections of the same tumour, it is reasonable toconclude that there is a continuous process produc-ing the inclusions.

It is interesting that 40 of our patients werefemales, for the prognosis from metastases ofmalignant melanoma of the iris is better in females.'7Is it possible, therefore, that these inclusions couldbe a pointer to a better prognosis in iris melanomas?

References

'Cowdry EV. The problem of intranuclear inclusions invirus diseases. Arch Pathol 1924; 18: 527-42.2Fischmann CF, Russell DS. The occurrence of intranuclearinclusions in culture of foetal leptomeninges. J PatholBacteriol 1940; 50: 53-9.3Zollinger HU. Beitrag zur pathogenese der einschluss-korper. Schweiz Z Allg Pathol 1951; 14: 446-55.4Kleinfeld R, Koulish S. Cytological aspects of mouse andrat liver containing nuclear inclusions. Anat Rec 1957;128: 443-5.5Leduc EH, Wilson JW. An electron microscopic study ofintranuclear inclusions in mouse liver and hepatomas.J Biophys Biochem Cytol 1959; 6: 427-30.6lwamoto T, Reese AB, Mund ML. Tapioca melanoma of theiris. Part 2. Electron microscopy of the melanoma cellscompared with normal iris melanocytes. Am J Ophthalmol1972; 74: 851-61.7Lommatzsch P, Klug H. Ein beitrag zur ultrastrukur desirismelanoms. Albrecht von Graejes Arch Klin Ophthalmol1977; 203: 101-17.8Pearse AGE. Histochemistry, Theoretical and Applied,3rd ed. London: Churchill 1968.9Leduc EH, Wilson JW. A histochemical study of intra-nuclear inclusions in mouse liver and hepatoma. J Histo-chem Cytochem 1959; 7: 8-16.

'0Serber BJ. Large nuclear inclusions in pituitary glandbasophils of golden hamster. Anat Rec 1961; 139: 345-52.

"Bouteille M, Kalifat SR, Delarue J. Ultrastructural varia-tions of nuclear bodies in human disease. J Ultrastruct Res1967; 19: 474-86.

1Sorbino-Sim6es MA, Conglaves V. Nuclear bodies inpapillary carcinomas of the human thyroid gland. ArchPathol 1974; 98: 94-9.

'3Ghadially FN. Ultrastructural Pathology of the Cell.London: Butterworths 1975: 50-78.

14Wessel W. Elektronemikroskopische untersuchungen vonintranuclearen einschlussk6rpern. Virchows Arch (PatholAnat) 1958; 331: 314-28.

"Sobel HJ, Schwartz R, Marquet MS, Passaic NJ. Non-viralnuclear inclusions. I. Cytoplasmic invaginations. ArchPathol 1969; 87: 179-92.

"Hogan MJ, Alvarado JA, Weddell JE. Histology of theHuman Eye. An Atlas and Text Book. Philadelphia: Saun-ders, 1971: 415.7Sunba MSN, Rahi AHS, Morgan G. Tumours of theanterior uvea: I. Metastasizing malignant melanoma of theiris. Arch Ophthalmol 1980; 98: 82-5.

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