Homogeneously staining regions (HSR) in a human malignant melanoma

Homogeneously Staining Regions (HSR) in a Human Malignant Melanoma Sonia Brieux de Salum, Irma Slavutsky, Santiago Besuschio, and Alberto Andino Pavlovsky

ABSTRACT: A case of nodular malignant melanoma (level V of Clark's classification) with homageneausly staining regions (HSR) an the long arm af one chromosome #2 is described. Ultrastructural observation of melanosomic and promelanosomic granules near Golgi's vesicles confirmed the histologic diagnosis. Chromosome analysis was performed on nine metaphases from a bone marrow sample and 76 metaphases from culture of the malignant skin tumor. Gbanding revealed the presence of a clone with trisomy #8 and another cell line with the HSR marker. This is the first report of HSR in human melanoma cells. As HSR has been found only in malignant cells, we believe that among the many factors that influence the patients' clinical evolution and poor response to treatment, the genic imbalance is of the utmost importance.

INTRODUCTION The results of c h r o m o s o m e b a n d i n g t e c h n i q u e s a p p l i e d to the s t u d y of h u m a n cancer and l e u k e m i a h a v e suggested a n o n r a n d o m n e s s of c h r o m o s o m a l changes in s o m e forms of h u m a n neoplasia, C h r o m o s o m a l changes o b s e r v e d in these studies h a v e s h o w n that specific marker c h r o m o s o m e s w e r e p r e s e n t in related and unrelated c a n c e r [1]. M a n y investigators h a v e f o u n d u n c o m m o n c h r o m o s o m e a b n o r m a l ities p r e s e n t in m a l i g n a n t m e l a n o m a . M o s t of the e x a m i n e d s p e c i m e n s w e r e metastatic cells, a l t h o u g h a n u m b e r of p r i m a r y t u m o r s h a v e also b e e n i n c l u d e d [2]. In this report, we p r e s e n t a case of m a l i g n a n t m e l a n o m a s t u d i e d d u r i n g relapse in w h i c h the c y t o g e n e t i c analysis r e v e a l e d the p r e s e n c e of c h r o m o s o m e # 2 w i t h a h o m o g e n e o u s l y staining region (HSR) in the long arm.

CASE REPORT G.A., a 66-year-old male, p r e s e n t e d in January 1979 w i t h an atypical p i g m e n t e d u l c e r a t e d lesion in his right leg. He was a d m i t t e d to the M i n i s t r y of E c o n o m y ' s Social H o s p i t a l and o p e r a t e d on in May 1979. At that time, the h e m a t o l o g i c data were: RBC 5,100,O00/mm 3, WBC 8300/ram 3, differential p o l y m o r p h o n u c l e a r s 68%,

From the Tissue Culture and Cytogenetic Department of the Hematologic Research Institute "Mariano R. Castex" (S. B. S.), National Academy of Medicine (S. B.), and Ministry of Economy's Social ttospital, (A. A. P.). Buenos Aires, Argentina. Address requests far Tejidos y Citogen~tica, Medicina, J. A. Pacheco Received August 13,

reprints to Dr. Sonia Brieux de Salum, Departamento de Cultivo de Instituto de Investigaciones Hematol6gicas, Academia National de de Melo 308l, 1425 Buenos Aires. Argentina. 1982; accepted March 14, 1983.

53 G 1984 by Elsevier Science Publishing Co., Inc. 52 Vanderbilt Ave., New York, NY 10017

Cancer Genetics and Cytogenetics 11. 53-60 (19841 0165-4608184/$03.00

54

s. Brieux de Salum et al.

lymphocytes 27%, monocytes 4%, eosinophils 1%, and basophils 0%. He presented with lymph node enlargement in the right inguinal region, and biopsy showed metastatic nodes, which were surgically excised, G.A. was administered dimethyl triazeno imidazole carboxamide (DTIC) 400 rag, and vincristine (Oncovin) 2 mg every 28 days (14 series). In March 1980, he relapsed with skin tumors, and at that time his lactate dehvdrogenase hormone (LDH) value was 5000 King. He was resistant to chemotherap~ and died in November 1980,

MATERIALS AND METHODS Pathologic Anatomy Biopsied material was fixed, dehydrated, and cleared as usual and then embedded in paraffin. Staining was done with hematoxylin-eosin and with Masson-Fontana to reveal the melanic pigment. Material from cell culture in the fifth passage was studied by electron microscopy. It was fixed with glutaraldehyde 3% in phosphate buffer from 2 hr and then washed with buffer for 24 hr. Postfixation was achieved with osmium tetroxide at 4°C for 2 hr. The material was dehydrated with alcohols, cleared with propylene oxide, and i n c l u d e d in a mixture of Araldite and propylene (1 : 1) at 4°C. Samples were kept at 65°C for 48 hr; then carved and cut in ultramicrotome. They were observed and photographed with a Siemens Elmiskop 1 electron microscope.

Cytogenetic Studies Chromosome analysis was performed on cells from metastatic skin tumors in April 1980 and in October 1980. Tissues were minced with scissors in a Petri dish containing F-10 m e d i u m with 15% fetal calf serum. Tissue fragments were placed in Falcon dishes with fetal calf serum. After 24 hr at 37°C, 7-8 ml of F-10 m e d i u m was added, and cell growth began after another 48 hr. Subculture was performed every 10 days. Mitotic cells were accumulated after five passages by exposure to Colcemid (0.1~xg/ml medium) for 3 hr before trypsinization and harvest. They were treated in a hypotonic 0.075 M KC1 solution for 30 rain at 37°C and fixed with methanolacetic acid (3:1) solution. A bone marrow sample from this patient obtained in May 1980 and studied by the direct method. Cells were also treated with a hypotonic 0.075 M KC1 solution for 30 min and fixed with methanol-acetic acid (3:1) solution: slides were air-dried. Chromosome preparations were stained with conventional Giemsa or banded with trypsin-Giemsa [3]. Identification of chromosomes according to the Paris nomenclature [4] was accomplished by using photographs of the metaphases.

RESULTS Macroscopic morphology of the sample proved to be of the nodular type of malignant melanoma, appearing as a bulky, dark, ulcerated nodule [5]. In the biopsied material, two cell types were microscopically observed: epithelioid and fusiform. Tissue invasion of the hypodermis places this melanoma into level V of Clark's classification [6] {Fig. 1).

HRS in Malignant Melanoma

55

Figure 1 Proliferation of melanoblasts of fusiform and epithelioid types, with production of melanic pigment (dark cells). Tumoral infiltration of the epidermis (right),

Homogeneous isolated granules (melanosomes) and small accumulations of crystalline structures with high electron density (promelanosomes) were observed with the electron microscope [7] (Fig. 2). Seventy-six metaphases from the malignant melanoma skin tumor culture were analyzed. Fifty-two came from the first culture and 24 from the second. Nine metaphases from bone marrow culture were also studied {Table 1). The modal chromosome n u m b e r was 46, with 25% pseudodiploid cells. Karyotype determination showed wide numerical variability of chromosomes from 40 to 45. While some cells were nullisomic for different chromosomes, there was clearly trisomy for others. Cells with more than 50 chromosomes did not form clones. G-banding technique showed HSR in the long arm of one chromosome #2 (35% of the cells) {Figs. 3 and 4) and a trisomy of #8 was observed in 20% of the rectaphases {Fig. 5). None of these cell types were present in the bone marrow sample.

DISCUSSION Many different chromosome anomalies have been detected in malignant melanomas 18 10]; recently, some authors have pointed to chromosome #1 as the most frequently involved in this pathology [1, 2]. In this study we present a patient with a nodular malignant melanoma, corresponding to level V of Clark's classification. U1trastructural observation of melanosomic and promelanosomic granules near Golgi's vesicles confirmed the histologic diagnosis of malignant melanoma. The clinical evolution of this patient was poor, and he hardly reacted to treatment. The modal chromosome n u m b e r of the patient's skin tumor culture was 46. with

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57

HRS i n M a l i g n a n t M e l a n o m a

Table I

N u m b e r of m e t a p h a s e s e x a m i n e d No. of chromosomes

Material analyzed First biopsy Second biopsy Bone marrow

<:40

40

41

42

43

44

45

46

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2 5 % p s e u d o d i p l o i d cells. T h o s e cells w i t h c o u n t s l o w e r t h a n 46 are n o t c o n s i d e r e d as s t e i n lines, b e c a u s e t h e r e is n o c o n s i s t e n t a b s e n c e or p r e s e n c e of s p e c i f i c c h r o mosomes. T h e c u l t u r e s e s t a b l i s h e d f r o m t w o t u m o r s a m p l e s of t h i s c a s e i n c l u d e d t w o k a r y o t y p i c a l l y d i s t i n c t p o p u l a t i o n s , o n e w i t h t r i s o m y # 8 , t h e s e c o n d w i t h a n abn o r m a l c h r o m o s o m e # 2 w i t h a n i n t e r s t i t i a l H S R o n 2q. O t h e r s t u d i e s i n m a l i g n a n t m e l a n o m a cell c u l t u r e s s h o w e d w i d e n u m e r i c a l v a r i a b i l i t y of c h r o m o s o m e s , ranging f r o m 24 to 50 c h r o m o s o m e s [2]. B a l a b a n - M a l e n b a u m [11] b e l i e v e s t h a t HSRs are large z o n e s w i t h h o m o g e n e o u s s t a i n i n g p r o p e r t i e s w i t h i n t h e c h r o m o s o m e , w h o s e s e c o n d a r y s t r u c t u r e differs f r o m

Figure 4 HSR on the long arm of chromosome #2 and normal chromosome #2, with diagrammatic presentation.

HSR on = 2

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HRS in Malignant Melanoma

59

normal (and, thus, is possibly less stable). HSRs have been found in malignant cells only; it is possible that they give malignant cells an advantage in overwhelming body defenses by means of a genetic imbalance that enhances tumoral growing ability. This e n h a n c e m e n t may result from an amplification m e c h a n i s m of those genes that stimulate tumoral viability [12], thus producing cells with proliferative and selective advantages. Alternatively, genic amplification could be associated with chromosome HSRs (stable phenotypes) or with double m i n u t e s (DMs, unstable phenotypes); the former are attached to chromosomes with stable centromeres, while the latter seem to lack a centromere [13]. DMs and HSRs are possibly alternative expressions of the same biologic entity [11]. This suggests that the breakdown of the HSR into DM happens during a cell replication cycle [14]. In the m e l a n o m a described here, we have only seen cells with HSRs in chromosome #2 and in hypodiploid, pseudodiploid, and diploid cells; no cells had DMs. This is the first report of HSR in h u m a n m e l a n o m a cells. The presence of HSRs in m u r i n e m e l a n o m a cell lines that developed resistance to methotrexate has been observed [151. In the present case, we believe that the malignant clone with the HSR marker was present from the onset of the disease, and it might be the determining factor in the clinical evolution of the patient and his poor response to treatment. An extra chromosome #8 was previously reported in this pathology [1] and recently in a metastatic malignant melanoma with 24 chromosomes [16]. It is worth remarking that, from the clinical point of view, there are many different factors to take into account w h e n determining a prognosis in melanomas: age and sex of the patient, site of the neoplasia, type and level of the invasion, thickness and extension of the lesion, presence or absence of metastasis in the lymphatic nodes of the region, and presence or absence of pigment in the tumor [7]. The association between certain chromosomal changes and some h u m a n neoplasias supplies a new tool to evaluate these pathologies from the diagnostic and prognostic, as well as the etiologic, points of view. It is, therefore, very important to introduce the systematic cytogenetic study of this pathology. This may become the principal element in the prognosis of the disease. Supported in part by grants from CONICET (Consejo Nacional de Investigaciones Cientificas y T6cnicas). The authors acknowledge the valuable technical assistance of Estela de Licen and Evelina Desset, and the photographic work of Osvaldo Miskoski.

REFERENCES

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