Supernumerary ring chromosomes containing chromosome 17 sequences

Supernumerary ring chromosomes containing chromosome 17 sequences

LEAD ARTICLE Supernumerary Ring Chromosomes Containing Chromosome 17 Sequences A Specific Feature of Dermatofibrosarcoma Protuberans? Florence Pedeuto...

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LEAD ARTICLE Supernumerary Ring Chromosomes Containing Chromosome 17 Sequences A Specific Feature of Dermatofibrosarcoma Protuberans? Florence Pedeutour, Jean-Michel Coindre, Gabriella Sozzi, Guido Nicolo, Agn/ s Leroux, Salvatore Toma, Monica Miozzo, Colette Bouchot, Frederick Hecht, Noel Ayraud, and Claude Turc-Carel

ABSTRACT: Mystery surrounds the mechanisms by which the u n c o m m o n cutaneous t u m o r dermatofibrosarcoma protuberans (DP) arises and progresses. A clue m a y be at h a n d in the form of extra abnormal chromosomes (one to three ring chromosomes p e r case with or without other chromosome abnormalities) seen in some 10 cases, including five cases in our experience. The specificity of the rings to DP would be enhanced if the rings were found to contain a contribution from a constant chromosome. We here report fluorescence in situ hybridization (FISH) results bearing on the chromosomal content of DP rings, together with clinical, pathologic, a n d cytogenetic documentation of our first two cases, which were briefly reported earlier, a n d three new DP cases. To dissect a translocation (in our 2nd case), we probed by FISH and discovered chromosome 17 sequences in the rings in all five DP cases. Nonfluorescent b a n d s were seen on some rings p a i n t e d with a whole chromosome 17 probe, indicating the presence in these rings of foreign chromosome sequences. The complexity of the rings was underscored by the detection in only one case of chromosome 17 centromeric sequences. The situation in DP seems to have parallels to that in well-differentiated liposarcoma, another t u m o r of intermediate malignancy with extra abnormal m a r k e r chromosome containing contributions from a constant chromosome and variable donors. In DP the s u p e r n u m e r a r y rings are c l e a r l y specific a n d significant.

INTRODUCTION The recognition of dermatofibrosarcoma protuberans (DP) goes back to 1924 w h e n Darier and Ferrand first reported this t u m o r in a p a p e r entitled "Dermatofibrosarcomas progressffs et r~cidivants ou fibrosarcomas de la peau" [Progressive recurrent dermatofibrosarcomas or skin fibrosarcomas} [1]. In the following year, 1925, Hoffmann further described this tumor and d u b b e d it ~Dermatofibrosarkoma protuberans" [2].

From the Laboratoire de C~n~ique MoMculaire des Cancers Humains (17.E, C. B., E H., N. A., C. T.-C.), URA CNBS 1462, Nice, France; Laboratoire cFAnatomopathologie (/.-M. C.), Fondation Bergoni~, Bordeaux, France; Divisione Oncologia Sperimentale A (G. S. M. M.), Istituto Nazionale Tumori, Milano, Italy; Laboratorio di Citoistologia Patologica (G. N.), Istituto Nazionale per la Bicerca sul Cancro, Genova, Italy; Labomtoire d/Anatomopathologie (A. L)., Centre Alexis Vautrin, Nancy, France; Istituto di Oncologia Clinica e Sperimentale (S. T.), Universit~ di Genova, Genova, Italy. Address reprint requests to: Claude Turc-Carel, Laboratoire de C~nggique Mo|~culaire des Cancers Humains, URA CNFiS 1462, Avenue de Valombrose, 06107 Nice, Cedex 02, France. Received November 22, 1993; accepted January 4, 1994.

© 1994 Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010

The names of Darier Ferrand a n d Hoffmann have subsequently b e e n u s e d in e p o n y m s for DP. Characteristically, DP arises in the deep dermis, w h e r e it presents as a firm n o d u l e or plaque. Favored locations are in the trunk a n d proximal segments of the limbs. It affects p r e d o m i n a n t l y adults in their young a n d m i d d l e years. Microscopy classically reveals a p r o m i n e n t cartwheel (storfform) pattern of cells. The histology need not be classical. Dermatofibrosarcoma protuberans is of intermediate malignancy, only rarely metastasizes, a n d w h e n it does, it is u s u a l l y late in the disease. However, DP grows in an infiltrative way a n d can r e c u r r e p e a t e d l y near w h e r e it began. The t y p e of cells in w h i c h DP starts is not k n o w n [3, 4]. The c h r o m o s o m e picture of DP was a complete b l a n k until 1990. Since then five DP cases w i t h c h r o m o s o m e abnormalities were reported [5-8]. There seems to be a single unifyhag feature to these cases, namely the presence of one or more supernumerary ring chromosomes. We have briefly reported two a d d i t i o n a l DP c a s e s (in a Letter to the Editor of Cancer Genetics a n d Cytogenetics [9]) a n d indicated that the rings in both cases contained c h r o m o s o m e 12 sequences. In this report we provide the details on these cases as w e l l a s three

Cancer Genet Cytogenet 76:1-9 (1994) 01654608/94/$07.00

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new DP cases with rings. The rings in all five cases contain chromosome 17 sequences.

MATERIALS AND METHODS

we used some slides for multiple hybridizations. After a first hybridization, slides were washed twice in 4 × SSC to remove mounting m e d i u m , then stored in ethanol 70% until denaturation u n d e r the same conditions as the first hybridization.

Patients The case reports of the five patients with DP are presented below, in the Results section. Clinical and pathologic features of these cases are s u m m a r i z e d in Table 1.

RESULTS

Cell Culture and Cytogenetics

tober, 1991 for recurrence of a cutaneous t u m o r over the sternum. In 1986, a 2-cm nodule has been excised from the same site and pathology had shown it to be a DP. The recurrence was p l u r i n o d u l a r and 7 x 3 cm in size. A w i d e resection was performed. Pathology showed two nodules (0.7 and I cm in size) d i a g n o s e d as DP and one nodule (2.5 cm in size) diagnosed as fibrosarcoma.

Cell cultures and standard cytogenetics were carried out as described previously [10-12]. Briefly, the specimens were received in the laboratory w i t h i n 1-2 days of surgery, submitted to mechanical and enzymatic dissociation, cultured in flasks or on glass coverslips in Petri dishes, and analyses done after 3-8 days in culture. Air-dried chromosome preparations were b a n d e d by standard techniques and results expressed according to the International System for Human Cytogenetic Nomenclature [13].

FISH Techniques Molecular Probes. The following probes were used for fluorescence in situ hybridization (FISH) on metaphase chromosomes: 1) Biotinylated a-satellite DNA probes (Oncor, USA) to chromosomes 7 (IT/Z1/DTZ2), 8 (D8Z1), 11 (D11Z1), 12 (D12Z3), 15 (D15Z1), 17 (D17Z1), 18 (D18Z1), X (DXZ1), and a shared probe to chromosomes 1 and 5 (DIZ7/D5Z1); 2) a biotinylated whole chromosome painting (WCP) DNA probe (Oncor) to c h r o m o s o m e 17; and 3) a direct-labeled spectrum orange WCP DNA probe (GIBCO-BRL, France) to chromosomes 1, 4, 12, and 17.

Hybridization and Detection. Hybridization and detection were performed according to Pinkel et al. [14] and to manufacturer's r e c o m m e n d a t i o n s with m i n i m a l modifications. Briefly, metaphase spreads from cells fixed in methanol:acetic acid (3:1) were stored in 70% ethanol at 4°C; slides were air-dried, treated with RNAse, and d e n a t u r e d by immersion in 70% formamide in 2 × SSC at 70°C. DNA was d e n a t u r e d for 5 minutes at 70°C, a p p l i e d to prewarmed slides, and incubated overnight at 37°C; after denaturation, probe DNA was hybridized to the target DNA on slides overnight in a moist chamber at 37°C. After sequential washes and detection with avidin-FITC (step omitted for the direct-labeled WCP probes from GIBCO), slides were e x a m i n e d for visualization of the fluorescent signal using a Zeiss A x i o p h o t microscope. Because we were working with a limited number of metaphases,

Patients Case 1 (T91125). A 59-year-old w o m a n was referred in Oc-

Case 2 (T91127). A sixth local recurrence of a t u m o r of the occipital scalp was resected from a 43-year-old man in November, 1991. The t u m o r was 6 x 5.5 x 13 cm and the pathologic diagnosis was a DP. A l t h o u g h a grade 2 malignant fibrous histiocytoma was also discussed, after peer review the diagnosis of DP infiltrating subcutaneous tissue but not bone was maintained. Histologically, the subcutaneous nodule was m a d e of s p i n d l e cells arranged in a storiform pattern. Tumor cells were relatively uniform w i t h a moderate mitotic activity (10 mitotic figures per 10 high-powered fields). The p r i m a r y t u m o r had been resected in March, 1978 and at that time was d i a g n o s e d as a neurofibroma. The same diagnosis was m a d e on the first (1982), second (1983), and third (1985) recurrences, but changed to DP after peer review. The mitotic index increased from the p r i m a r y t u m o r to the third recurrence. The fourth (1986) and fifth (1988) recurrences were also diagnosed as DP.

Case 3 (T92338). In December, 1992, a 62-year-old m a n underwent a diaphragmatic and extraparietal m u s c u l a r resection i n c l u d i n g the 10th and 11th ribs, in a d d i t i o n to a large surgical excision of an 8 x 5 × 5-cm t u m o r for the third recurrence of a DP. Histologically, the t u m o r showed a spindle-cell sarcoma with a focal storiform pattern, some sclerous modifications, and numerous vessels simulating the pattern of a hemangiopericytoma. The cells had low nuclear p l e o m o r p h i c but high mitotic activity (more than 20 mitosis/10 high-powered fields). The diagnosis of the primary lesion seated in the l u m b a r area, resected in 1958, was not known. The diagnosis of DP was m a d e on the first recur-

Table 1 Clinicopathologic characteristics of 5 dermatofibrosarcoma protuberans cases Case no.

Laboratory identification

Sex/age (yr)

Site

Primary tumor Year of diagnosis

1 2 3 4 5

T91125 T91127 T92338 PM T93456

F/59 M/43 M/62 F/34 F/68

Presternal skin Occipital skin Lumbar skin Right dorsal skin Right inguinal skin

1986 1978 1958 1973 1993

Primary/recurrence Recurrence Recurrence Recurrence Recurrence Primary

No. of the recurrence 1 6 3 2

Specificity of Chromosome 17 Rings in Dermatofibrosarcoma

3

rence in 1985. A second recurrence occurred in June, 1988. The infiltrating tumor, w h i c h was s u r r o u n d e d by satellite nodules, was resected together with the 12th rib, overlying muscular wall and l u m b a r skin. The resection was extended 2 months later by a w i d e thoracolumbar pariectomy showing some residual tumoral foci seated in the dermis at some distance from the prior resection margin. The diagnosis of these two resections was again DP. Case 4 (PM). A 34-year-old woman presented in August, 1992 with a superficial 5 × 6-cm mass in the right dorsal region. She had u n d e r g o n e surgery for a primary DP at the same site 19 years before and for a local recurrence 5 years later. Extended t u m o r resection was undertaken and histopathologic examination revealed local recurrence of DP. Histologically, the tumor showed the presence of spindle-shaped cells arranged in a diffuse storiform pattern. The overlying e p i d e r m i s was intact whereas the dermis and subcutaneous fat tissue were massively infiltrated by tumor. The resected m a r g i n s were free of disease. Case 5 (T93456). A 68-year-old woman was referred in April, 1993 for a t u m o r of the right inguinal region w h i c h she said had a p p e a r e d 4 months earlier. On examination, there was a 5-cm m u l t i n o d u l a r cutaneous and subcutaneous tumor. A w i d e local excision was performed. Pathologic examination revealed a DP w i t h diffuse infiltration of dermis and subcutis by s p i n d l e cells arranged in a distinct storiform pattern (Fig. 1). S p i n d l e cells were uniform with little nuclear p l e o m o r p h i s m and a low mitotic activity (five mitotic figures per 10 high-powered fields). Cytogenetic A n a l y s i s Case I (T91125). This case, w h i c h was briefly reported earlier [9], had 20 RHG-banded metaphases analyzed after 3 days in culture: 11 cells w i t h a normal 46,XX karyotype and 9 cells w i t h 49,XX, + 5, + 8, +r (Fig. 2).

Case 2 (T91127). This case, w h i c h was also briefly reported earlier [9], had 21 RHG-banded metaphases analyzed after 7-8 days in culture: eight cells w i t h a normal 46,XY karyotype, 10 cells w i t h 46,XY, + der (5) t(5;13) (p11;q11), - 13, add (17)(p13), - 18, + r (di and tricentric) (Fig. 3), and two cells with 47, idem, + r. A variant cell showed a derivative chromosome 12 (add(12)(p13)) in a d d i t i o n to the rings. Case 3 (T92338). This new case had 20 RHG-banded metaphases analyzed after 6 days in culture: two cells with 47, XY, - 4, - 22, + rl, + r2 x 2 (where r l was larger than r2), 14 cells with 48, idem, + mar (where mar was a small unidentified marker) (Fig. 4), three cells with 49,idem, + 2mar, and one variant cell identical to the m a i n l i n e showing two normal chromosomes 4. Case 4 (PM). This new case had 12 GTW-banded metaphases analyzed after 7 days in culture: 10 cells w i t h 50, XX, + 4, + 5, + 8, + r (Fig. 5), and two cells w i t h 51,idem, + r. Case 5 (T93456). This case, also new, had 12 RHG-banded metaphases analyzed after 6 days in culture and showed a

Figure 1 Typical aspect of DP characterized by short fascicles of slender spindle cells arranged in a storiform (cartwheel) pattern (case 5, H&E, × 64).

modal number 92 with some consistent numerical and structural chromosome abnormalities, i n c l u d i n g rings, in about half of the cells. The composite karyotype was: 74-95, XXXX, - 2 , - 3 , - 4 , - 5, +Z -10, -10, +11, + 11, +12, +12, -15, -19, - 20, - 20, + r, + r, + marl, + mar2, + mar3, + mar4, + d m i n (Fig. 6). FISH A n a l y s i s

Case 1 (T91125). The origin of the centromere of the ring r e m a i n e d obscure in this case. No signal was obtained with biotinylated centromeric probes to chromosomes 1, 5, 7, 8, 12, 17 (Fig. 7a), 18, or the X. Nor was signal seen with WCP DNA probes to chromosomes 1, 2, and 11. The WCP probe to chromosome 17 showed narrow alternating fluorescent and nonfluorescent bands on the ring (Fig. 7b). Case 2 (T91127). The centromeric probe to chromosome 17 showed two or three signals on the ring chromosome (Fig. 7c). Contrary to the statement in our brief c o m m u n i c a t i o n [9] indicating that with a WCP probe to chromosome 17 rings were "painted homogeneously" the ring chromosome in most of the metaphases showed alternation of fluorescent and nonfluorescent bands (Fig. 7d). WCP probes to chromosomes 1 and 12 gave no signals on the rings. With centromeric probes to chromosomes i and 5, we confirmed the presence of the chromosome 5 centromere in the derivative, der(5) t(5;13)(p11;q11). Case 3 (T92338). Of the three rings present in all metaphases, only the two smaller rings showed a positive fluorescent signal with the WCP DNA probe to chromosome 17 (Fig. 7e). The large ring showed no hybridization with a WCP probe to chromosome 4 (which seemed a logical candidate because it was m o n o s o m i c in all but one cell). None of the rings was positive with a WCP probe to chromosome 12 or a chromosome 17 centromeric probe. Case 4 (PM). The rings in this case hybridized with the WCP 17 probe, except in a small (centromeric ?) region (Fig. 7f,g). The centromeric probe to c h r o m o s o m e 17 gave no signal.

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Case 5 (T93456). The large ring c h r o m o s o m e in this new case showed a striped pattern w i t h the WCP 17 probe (Fig. 7h,i) and was negative with the c h r o m o s o m e 12 WCP and c h r o m o s o m e 17 centromeric probes. DISCUSSION We performed c h r o m o s o m e and FISH studies on five cases of DP ([9] and this report). Each case had at least one or more

s u p e r n u m e r a r y ring chromosomes, as d i d all five cases of DP reported, to our knowledge, with chromosome abnormalities in the literature [5-8]. In three of the cases in the literature the ring was the only detectable c h r o m o s o m e aberration [6, 8]. The consistent presence and sometimes solitary nature of the s u p e r n u m e r a r y ring strongly suggests that it may represent a p r i m a r y cytogenetic event in DP. Seven of the 10 DP cases have h a d other c h r o m o s o m e abnormalities aside from the rings. To take the addition of chro-

Specificity of Chromosome 17 Rings in Dermatofibrosarcoma

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J F i g u r e 3 Supernumerary ring chromosome (arrow) in a full R-banded karyotype from case 2. The small arrowhead indicates the derivative 17 chromosome and the large arrowhead the derivative t(5;13). Loss of chromosome 18 is clonal.

m o s o m e material, the case of Bridge et al. [5] had trisomy 8; the case of Stephenson et al. [7] h a d trisomy 7, 11, 13, 14, and 15; our case I had trisomy 5 and 8; our case 2 had partial trisomy 5 due to a der(5)t(5;13)(pll;q11); our case 4 had trisomies 4, 5, and 8; and our case 5 had supernumerary chrom o s o m e s 7, 11, and 12 in a near-tetraploid context. Of these 15 instances of the a d d i t i o n of identifiable rod c h r o m o s o m e material, there was recurrent involvement of chromosome 5 (three cases), c h r o m o s o m e 7 (two cases), c h r o m o s o m e 8 (three cases), and chromosome 11 (two cases) (Table 2). Thus, four chromosomes (numbers 5, 7, 8, and 11) account for 10/15 (67%) of the extra rod chromosome material. The non-random

cytogenetic changes in DP may well go beyond the presence of a s u p e r n u m e r a r y ring. Using WCP probes, we found that in all cases the supern u m e r a r y ring chromosomes contained chromosome 17 sequences, as we have briefly described for cases I and 2 [9]. In only one case (case 2) d i d a ring contain chromosome 17 u-satellite centromeric sequences. That ring a p p e a r e d to be a tricentric ring of chromosome 17. In the other cases, these pericentromeric sequences were absent. This observation, together with the fact that in cases 1, 2, 3, and 5 the fluorescent pattern showed an alteration of painted and u n p a i n t e d bands, calls to m i n d the similarity between these structures

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Figure 4 Three extra ring chromosomes (small arrowhead for the large ring, large arrowheads for the two others) and a small marker (arrow) in a full karyotype of a metaphase from the mainline of case 3. Losses of chromosomes 4 and 22 are clonal.

and those we have described in supernumerary ring and large rod marker chromosomes in well-differentiated liposarcomas (WDLPS} [15, 16]. The striped pattern indicates that chromosomes other than 17 are involved in the constitution of the ring. If observed in a larger number of cases, supernumerary ring chromosomes containing chromosome 17 sequences may constitute a specific element of DP. This new element might help to make the diagnosis, w h i c h is sometimes difficult and complex to establish. Our case 2 illustrates some

of these difficulties. The p r i m a r y t u m o r and the first three recurrences, all located on the scalp, were initially diagnosed as neurofibromas and then revised to DP. As a matter of fact, the differential diagnosis between DP and diffuse neurofibroma located in the head and neck raises particular problems for the pathologist [3, 17]. In case 2, the sixth recurrence (a s p e c i m e n of w h i c h was submitted to us), the diagnosis of grade 2 malignant fibrous histiocytoma was also advocated, but it was finally m a i n t a i n e d as DP. According

Specificity of Chromosome 17 Rings in Dermatofibrosarcoma

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Figure 5 The arrows indicate the extra-ring chromosome and the supernumerary chromosomes 4, 5, and 8 in a full G-banded karyotype from case 4.

to Enzinger and Weiss [3], subcutaneous DP is difficult to distinguish from benign histiocytofibroma, and deep infiltrating DP is hard to distinguish from malignant fibrous histiocytoma (MFH}. Moreover, as DP recurs (and there may be numerous recurrences}, DP can become more and more similar to fibrosarcoma [3]. Compounding the pathologic difficulties is the controversial classification of these tumors. DP is for the m o m e n t classified a m i d the histiofibrocytic tumors w i t h a degree of m a l i g n a n c y intermediate between that of benign and malignant fibrohistiocytomas. However, this classification is not universally agreed upon, and a neural origin hypothesis is not excluded [3]. Some m o l e c u l a r and

cytogenetic data are available for fibrohistiocytic tumors [18-20]. Like DP, MFH can exhibit ring chromosomes. We have observed that the rings from two of our MFH cases were not painted with a WCP 17 probe [unpublished observations), but in one of these cases was painted w i t h the WCP 12 probe. We showed that chromosome 17 sequences were always present in the rings of our five DP cases. Chromosome 12 sequences were not seen, at least in the three cases tested. Therefore, it may prove that chromosome 17 sequences in the extra ring chromosomes constitute a specific feature of DP allowing their separation from MFH. The rings of the two MFH cases m e n t i o n e d above were seen in the context of a

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mar 1 Figure 6

mar 2

mar 3

mar 4

dmin

Ring and marker chromosomes and dmin found in RHG-banded metaphases from case 5.

and low-grade MFH, although seeming superficially to share an identical chromosomal profile, are distinct entities. Three arguments are in favor of a neural origin hypothesis for DP: 1) a small proportion (about 5%) of DP cases contain a population of dendritic cells producing melanin (Bednar tumors) [3, 21-23]; 2) ultrastructural studies of DP show cells with Schwann cell features [3]; 3) antigen CD-34 is ex-

very complex karyotype. The tumors were of high grade. Whether rings were found with s i m p l e r karyotypes in lowgrade MFH contain chromosome 17 sequences has to be investigated. If they do, it w o u l d call for the search of shared chromosome 17 rearrangements at the origin of their identical clinical outcome. If low-grade MFH rings are negative for chromosome 17 sequences, this w o u l d indicate that DP

Figure 7 Fluorescence in situ hybridization with WCP or a-satellite centromeric probes to chromosome 17 on partial metaphases from the five DP cases. In all partial metaphases, large arrowheads and small ones point, respectively, to normal chromosome 17 and ring chromosome(s). The lack of hybridization of the R-satellite centromeric probe to chromosome 17 is illustrated on the ring from case 1 (a). Ring from case 2 showed three positive signals with the previous centromeric probe (c). The fluorescence pattern of the rings using the spectrum-orange WCP probe to chromosome 17 is shown in cases 1, 2, 3, 4, and 5 (b, d, e, f, h, respectively). The large ring from case 3 was not painted (e). DAPI-stained partial metaphases from cases 4 and 5 (g,i).

A a "4



9

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S p e c i f c i t y of Chromosome 17 Rings in Dermatofibrosarcoma

Table 2

Chromosomes f o u n d to be present as s u p e r n u m e r a r y i n addition to the ring chromosome in the 5 previously published cases and the 5 cases in this report

Case reports (references) Bridge et al., 1990 [5] Mandahl et al., 1990

Supernumerary chromosomes in addition to the ring

9.

10.

8 None

[6] Stephenson et al., 1992 [7] (3rndal et al., 1992 [8], cases 4 and 5 This report Case 1 Case 2 Case 3 Case 4 Case 5

9

11. 7, 11, 13, 14, 15 None

5,8 der 5 p mar 4, 5, 8 7, 11, 12

12.

13.

14.

15. pressed in DP cells, a n d not in b e n i g n fibrous histiocytomas [24]. It may be noteworthy that among the genes m a p p e d to chromosome 17 is the NF1 gene in 17q11.2, involved in neurofibmmatosis 1 [25, 26], but there is no evidence yet that N F / i s involved in DE Whether molecular rearrangement occurs in the N F / g e n e in DP as a result of ring formation should be studied further.

16.

17. 18.

This work was supported by CNRS and grants from CRAMTS, Marseille, the Association pour la Recherche sur le Cancer (ARC-Villejuif), the F~d~ration Nationale des Centres de Lutta contre le Cancer, France, the Italian National Research Council (special project ACRO), and the Italian Association Cancer Research (IARC).

19.

20. REFERENCES 1. Darier J, Ferrand M (1924): Dermatofibrosarcoma progressifs et r~cidivants ou fibrosarcomas de la peau. Ann Dermatol Syph 5:545-548. 2. Hoffmann E (1925): Uber das knollentreibendeFihrosarkom der Haut (Dermatofibrosarkoma protuberans). Dermat Ztschr 43:1. 3. Einzinger FM, Weiss SW (1988): Soft Tissue Tumors, 2nd ed. St Louis, The CV Mosby Company. 4. Fletcher CDM, McKee PH (1990): Pathobiology of Soft Tissue Tumors. Edinburgh, Churchill Livingstone. 5. Bridge JA, Neff JR, Sandberg AA (1990): Cytogenetic analysis of dermatofibrosarcoma protuberans. Cancer Genet Cytogenet 49:199-202. 6. Mandahl N, Heim S, Willen H, Rydholm A, Mitelman F (1990): Supernumerary ring chromosome as the sole cytogenetic abnormality in a dermatofibrosarcoma pmtuberans. Cancer Genet Cytogenet 49:273-275. 7. Stephenson CE Berger CS, Leong SPL, Davis JR, Sandberg AA (1992): Ring chromosome in a dermatofibrosarcoma protuberans. Cancer Genet Cytogenet 58:52-54. 8. (3rndal C, Mandahl N, Rydholm A, Willen H, Brosj6 O, Heim S, Mitelrnan F (1992): Supernumerary ring chromosomes in five

21. 22.

23.

24.

25.

26.

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