ANTICANCER RESEARCH 24: 19-26 (2004)

CGH Analysis Shows Genetic Similarities and Differences in Atypical Fibroxanthoma and Undifferentiated High Grade Pleomorphic Sarcoma

DANIELA MIHIC-PROBST1*, JIANMING ZHAO1*, PARVIN SAREMASLANI1, ANGELA BAER1, CHRISTIAN OEHLSCHLEGEL2, BRUNO PAREDES3, PAUL KOMMINOTH4 and PHILIPP U. HEITZ1

1Department of Pathology, University Hospital, Zürich; 2Institute of Pathology, Cantonal Hospital, St. Gallen; 3Department of Dermatology, University Hospital Bern; 4Institute of Pathology, Cantonal Hospital, Baden, Switzerland

Abstract. Background: Atypical fibroxanthoma (AFX) and hyperchromasia and pleomorphism. Mitotic figures, including undifferentiated high grade pleomorphic sarcoma (UpS) are abnormal forms, are common. In contrast to UpS, AFX is histologically very similar, if not identical. However, they differ located in the dermis, lacks significant subcutaneous infiltration significantly in clinical outcome. Materials and Methods: We and has an excellent prognosis after complete surgical excision. used comparative genomic hybridization (CGH) to screen 24 It should be noted, however, that in exceptionally rare cases a AFX and 12 UpS for genomic alterations. Results: DNA copy lesion qualified as AFX can behave in an extremely aggressive number changes were observed in 20/24 AFX and in all UpS. manner, comparable with that of UpS (1, 2). AFX is a solitary The most frequent alterations occurring with comparable tumor usually occurring on sun-damaged skin of the head and frequency in both tumors were deletions on chromosomes 9p neck of elderly persons. Helwig first described 20 tumors in and 13q. We also detected statistically significant differences of 1963 (3, 4). Solar UV- induced p53 mutations may play an genetic alterations between the two tumors concerning important role in the pathogenesis of AFX (5, 6). deletions on 1q, 3p, 5q, 11p, 11q, gains on 7q, 12q and high The terms UpS and pleomorphic malignant fibrous level gains on 5p and 11q. Conclusion: Despite their very histiocytoma are used synonymously in the literature (7). similar histology, AFX and UpS show clear differences in their However the validity of the latter has been questioned for two genetic alterations. This might contribute to the different main reasons: 1) the introduction of immunohistochemistry biological behavior of the two tumors. On the other hand the has shown a definable line of differentation in most high grade similarities in genetic alterations on chromosomes 9p and 13q pleomorphic sarcomas (8); 2) undifferentiated high grade might suggest a common pathogenetic pathway. pleomorphic sarcomas show no evidence of histiocytic differentiation. The histology of atypical fibroxanthoma (AFX) and Several studies using various approaches, e.g. ploidy undifferentiated high grade pleomorphic sarcoma (UpS) is analysis by flow cytometry (9) and image analysis (10), failed virtually identical: it is characterized by a mixture of large to elucidate the pathogenetic pathway of AFX and UpS (11- atypical giant and spindle cells with considerable nuclear 13). Further attempts to differentiate AFX from UpS on the basis of proliferation (14), apoptosis and regulators thereof, i.e. p53 and bcl2 (15), as well as immunohistologic studies were not successful (16). This has led to the hypothesis that *Contributed equally to this study AFX may represent a superficial form of UpS (17). However, a recent report showed a different expression Correspondence to: Daniela Mihic-Probst, M.D., Department of of the LN2 antigen (CD74) in the two lesions: while ninety Pathology, University Hospital, Schmelzbergstrasse 12, 8091 percent of UpS stained strongly for LN2, this reaction was Zürich, Switzerland. Tel: +41-1-255-2595, Fax: +41-1-255-4440, e- absent in the vast majority of AFX (18). mail: [email protected] To date, a small number of chromosomal analyses of UpS Key Words: Atypical fibroxanthoma, undifferentiated high grade have been carried out (19-22) but, to our knowledge, only pleomorphic sarcoma, pleomorphic malignant fibrous one detailed chromosomal analysis including AFX, focusing histiocytoma, comparative genomic hybridization. on ras-mutations, has been reported (23).

0250-7005/2004 $2.00+.40 19 ANTICANCER RESEARCH 24: 19-26 (2004)

In the present study, we used comparative genomic CGH analysis. CGH allows a rapid detection of DNA copy number hybridization (CGH) to screen for genetic alterations in two changes in the entire genome, providing an overview of genomic groups of tumors; i.e. 24 atypical fibroxanthomas (AFX) and imbalances in a given tumor. CGH was performed essentially as previously described (25). 12 undifferentiated high grade pleomorphic sarcomas One microgram of tumor DNA was labeled with Spectrum (UpS). Phenotypic differentiation of UpS was excluded. Green-dUTPs (Vysis, Downers Grove, IL, USA) by nick The goals of this study were: 1) to screen for frequently translation (BioNick kit, Life Technologies, Basel, Switzerland). altered chromosomal regions in AFX and UpS, 2) to Spectrum Red-labeled normal reference DNA (Vysis) was used elucidate molecular similarities and/or differences of the for co-hybridization. The hybridization mixture consisted of 200 two tumors, and 3) to provide a molecular basis for the ng Spectrum Green-labeled tumor DNA, 200 ng of Spectrum similarity in morphology of the two tumors on the one hand Red-labeled normal reference DNA and 10-20 Ìg of human Cot- 1 DNA (Life Technologies) dissolved in 10 Ìl of hybridization and of their different biological behavior on the other. buffer (50% formamide, 2X SSC, pH 7.0). Hybridization took place over 3 days at 37ÆC to sex-matched normal human Materials and Methods metaphase spreads (Vysis). Slides were washed at 45ÆC three times for 10 min in wash solution (50% formamide, 2X SSC, pH Patients and tumor specimens. Specimens of thirty-six patients (28 7.0) followed by twice for 5 min in 2X SSC. The chromosomes men and 8 women, age 39 to 97 years) were selected for this study. were counterstained with 4,6-diamidino-2-phenylindole (DAPI) Twenty-four patients suffered from AFX, twelve from UpS for identification. (Clinical data of the patients: Table I). None of the patients had Digital images were collected from six to seven metaphases received chemotherapy or radiotherapy prior to surgery. Histology using a cooled charge-coupled device (CCD) camera of all tumors was reviewed by one pathologist (D.M.). Histological (Microimager 1400; Xillix Technologies, Vancouver, Canada) evaluation and classification of the tumors were based on attached to a Zeiss Axioskop microscope and a computer. The established criteria (17, 7). software program QUIPS (Vysis) was used to calculate average All AFX were located in the dermis without infiltration of green-to-red ratio profiles for each chromosome. At least six subcutaneous tissue or necrosis. Only tumors with abundant observations per autosome and three observations per sex atypical pleomorphic cells and atypical mitoses were observed. chromosome were included in each analysis. Chromosomal gains Immunohistochemically they sometimes showed focal positivity for and losses were defined as fluorescence ratio values above 1.2 antitrypsin, antichymotrypsin and CD 68. Other types of neoplasms or below 0.8, respectively. Amplification was assumed at were excluded by lack of immunostaining for cytokeratins, chromosomal regions where the green to red ratio exceeded 1.5. epithelial membrane antigen, S-100 protein, desmin, ·-smooth Since some false-positive results were found in normal tissues at muscle actin and CD 34 (Fig. 1A, B, C). 1p32-pter, 16p, 19 and 22, gains at these G-C-rich regions were Seven UpS were localized in the subcutis while five were found excluded from all analyses. within skeletal muscle. Two of the subcutaneous tumors showed involvement of the adjacent dermis (Table I ). The study comprised Statistics. For scoring of genetic alterations, whole chromosome exclusively UpS not showing any definable line of differentiation. changes were scored as one event. All other changes were scored The tumors were extensively sampled and phenotyping resulted in by chromosomal arm. A loss and a gain on one arm were scored as a lack of immunoreactivity for desmin, ·-smooth muscle actin, two changes, whereas two separate losses (or gains) on the same myogenin, cytokeratins, CD34 and S-100 protein in all tumors arm were scored as one change. (Figure 1D, E, F). Contingency table analysis was used to compare frequencies of genomic alterations in different groups of tumors. All statistical DNA extraction. All examined tumor specimens were formalin-fixed tests were two-sided and were considered to be statistically and paraffin-embedded. Representative tumor samples were significant at p<0.05. microdissected and scraped from five to ten 10-Ìm-thick unstained tissue sections under a stereomicroscope (Stemi DV4, Zeiss, Results Germany) at 10 to 40x magnification. Genomic DNA was isolated DNA copy number changes were observed in 20/24 AFX as previously described (24). Briefly, representative tumor material and in all 12 UpS (Table I ). UpS showed a statistically was collected in a 1.5 ml Eppendorf tube containing 100% ethanol and centrifuged to the bottom of the tube (5 min at 14000 rpm). significant (p value < 0.0009) larger number of genomic After the ethanol had been removed, the tissue material was alterations than AFX. The average number of alterations treated three times with 1 ml of fresh xylol for 15 min at 55ÆC in a per tumor was 3.3±0.7 in AFX vs. 8.9±1.1 in UpS (Table thermomixer. Subsequently, the specimens were treated with 100% II). High-level gains (amplifications) were observed in 9/12 ethanol and centrifuged for 10 min at 14000 rpm. After a second UpS. By contrast, only 1/24 AFX showed amplifications. treatment with 70% ethanol and subsequent centrifugation, the The chromosomal regions with DNA copy number tissue material was dried in a speed-vac for 10 min at room alterations (losses and gains) identified by CGH are shown temperature. The tissue was digested overnight with 0.5 mg/ml Proteinase K (Sigma, St. Louis, MO, USA) in 1ml digestion buffer in Figure 2, Figure 3 and Table III. The most common (50 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.5% SDS) at 50ÆC. The DNA copy number changes were losses on 9p and 13q. DNA was purified by standard phenol-chloroform extraction and These were found in a very similar ratio in both tumor ethanol precipitation and quantified by spectrophotometry (24) . types: AFX (9p: 54%; 13q: 42%) vs. UpS (9p: 58%; 13q:

20 Mihic-Probst et al: CGH Analysis in Atypical Fibroxanthoma

Figure 1. Histology of atypical fibroxanthoma (AFX: A, B, C) and undifferentiated high grade pleomorphic sarcoma (UpS: D, E, F). A. Dermal localization of AFX. Haematoxylin-eosin x 16. B. Atypical spindle and giant cells in AFX with considerable nuclear pleomorphism and mitotic figures. Haematoxylin- eosin x 200. C. Lack of staining for a-smooth muscle actin (note a small vessel as internal positive control). Immunohistochemical stain and haematoxylin counterstain x 200. D. Intramuscular localization of UpS. Haematoxylin-eosin x 16. E. Similiar histology to AFX (B) of UpS. Haematoxylin-eosin x 200. F. Lack of staining for CD 34 (note a small vessel as internal positive control). Immunohistochemical stain and haematoxylin counterstain x 200.

58%). Other areas containing less common alterations 28). Our finding of deletions on chromosomes 9p and 13q included Y- (8%), 12p+ (16.6%), 1q-, 4q-, 5q-, 6q-, 10q-, in UpS is in accordance with results of one previous CGH 11q-, 18q-, 4q+, 7q+, 8q+, 12q+ (each 25%), 3p-, 11p-, study on UpS (19). 11q+ (each 33%), and 5p+ (42%) in UpS and 3p-, 10q-, We found genetic alterations in 20/24 AFX. The most 11p-, 4q+, 5p+ 8q+ (each 4%), 4q-, 6q-, 18q- (each 8%) frequent deletions involved chromosomes 9p and 13q and and Y- (25%) in AFX. The difference between the ratios the ratios in AFX were similar to that in UpS. Our results in the two tumor types reached statistical significance at therefore demonstrated some potentially important genetic the following loci: 1q-, 3p, 5q-, 11p-, 11q-, 5p+, 7q+, similarities between AFX and UpS. However, we also 11q+, 12q+. Amplifications were observed in UpS on 4q, detected statistically significant differences of genetic 5p, 7q, 11q, 12p, but in only one AFX (on 9p). alterations between the two tumors concerning deletions on 1q, 3p, 5q, 11p, 11q, gains on 7q, 12q, and high level gains Discussion on 5p and 11q. Some genes located in these chromosomal regions might contribute to the different biological behavior To our knowledge, only one analysis of AFX at the DNA of the two tumors. level, focusing on ras-mutations, has been published to date A recent study (23) has disclosed H-ras (11p) and K-ras (23). Therefore, our data can mainly be compared to (12p) mutations in UpS, which were shown to be absent in previously published molecular studies on UpS (19-22, 26- AFX. The authors postulated that this difference may be

21 ANTICANCER RESEARCH 24: 19-26 (2004)

Table I. Clinical and genetic data of 12 undifferentiated high grade pleomorphic sarcoma (UpS) and 24 atypical fibroxanthomas (AFX).

Age/ Local Metastasis CGH findings No. Sex Diagnosis Location recurrence Gains Loss

1 81/f UpS, G3 Thigh, sc. yes nk 7q32-qter, 8q, 9q31-qter 6q, 9p, 11p, 11q-23ter, 12p, 13q, 18q, xq21-qter 2 55/m UpS, G3 Calf, s.m. yes nk 5pcen-p14, 20q12 3q, 5q, 8p, 9p, 13q, 14q, 18q 3 * 63/m Ups, G3 Forearm, sc. yes yes 5qcen-q12 4p, 4q, 9p12-p21.1, 13q 4 97/m UpS, G3 Calf, sc. nk nk 8q, 9q13-q22, 11qcen -q22, 1q31-qter, 3p12-p21, 12q14-qter, 17q21-qter, 18p, 5p14-p15, 5q23-qter, 18qcen-q12 11q23-qter, 13q, 21q 5 63/m UpS, G3 Forearm, sc. yes yes 17p12-p13, 17q22-qter 4pcen-p15.2, 4q, 9p12-p21.1, 13q 6 82/m UpS, G3 Hand, sc. yes nk 7q, 8q, 11q14-q22 3p, 3q, 4q33-qter, 9p, 10p, 10q, 13q31-qter, 18q21.2-qter 7 39/m UpS, G2 Forearm, sc. nk nk 4q28-32, 11q13-qter, 21q, 6q23-qter, 11p Xp, Xq 8 88/f UpS, G2 Calf, s.m. yes yes 5pcen-p14, 6q21-q26, 12p, 9pcen-p21, 11q22-qter 12qcen-q21 9 64/m UpS, G3 Mandible, s.m. nk. nk 4pcen-p14, 4qcen-q32, 1q32-qter, 6q23-qter, 11p 5pcen-p14, 11q13-qter, 13q32-qter, 21q, Xp, Xq 10 55/m UpS, G3 Calf, s.m. nk nk 2q22-q32, 5pcen-p14 None 11 70/m UpS, G2 Thorax, s.m. ** nk 1pcen-p31, 7q21-q31, 8q22-qter, 3p21-pter, 10q24-qter, 12qcen-q21, 14q13-q24 16p, 16q, Y 12 75/m Ups, G2 Back, sc. nk nk 1q22-q24, 4q12-q24, 1q32-qter, 2p22-pter, 2q, 5pcen-p14, 15q15-25, 17p 3p14-p21, 5q32-qter, 9p, 10q,11p, 13q, 16q 13 43/f AFX Head, s.d. nk nk None None 14 49/m AFX Head, s.d. nk nk None Y 15 94/f AFX Neck, s.d. nk nk 3p, 3q, 4p, 4q12 4q21-qter, 9p, 9q, 13q, 16q 16 87/m AFX Ear, s.d. nk nk 2p, 2q, 9q32-qter, 17p, 4p, 4q, 9p, 11p, 17q, 20q, Xp 13q12-q14, 14qcen-q13 17 85/m AFX Head, s.d. nk nk None 6q, 7p, 7q, 9p, 13q, Y 18 76/f AFX Head, s.d. nk nk None 9p, 10p, 13qcen-q31, Xq21-qter 19 75/m AFX Ear, s.d. nk nk None 13q21-qter, 18p, 18q, Y 20 81/m AFX Head, s.d. nk nk None 13q 21 77/m AFX Head, s.d. nk nk 1q None 22 46/m AFX Thorax, s.d. nk nk None 2q34-qter, 6p, 6qcen-q15, 9p, 10p, 10q, 16q 23 51/f AFX Head, s.d. nk nk 3q22-qter, 8q13-q21, 3p 9p21-pter, 11p12-p13 24 68/m AFX Head, s.d. nk nk None None 25 87/m AFX Head, s.d. nk nk None 9p, Y 26 85/f AFX Head, s.d. nk nk None 9p 27 75/f AFX Head, s.d. nk nk None 9p, 9q 28 75/m AFX Ear, s.d. nk nk None None 29 80/m AFX Head, s.d. nk nk None 9p, 9qcen-q21, 13q 30 77/m AFX Head, s.d. nk nk None 9p, 9qcen-q33, 13q 31 73/m AFX Head, s.d. nk nk None 18q 32 67/m AFX Head, s.d. nk nk None Y 33 89/m AFX Head, s.d. nk nk None None 34 72/m AFX Head, s.d. nk nk None 9p, 13q, 16p, 16q 35 69/m AFX Head, s.d. nk nk None 9p, Y 36 89/m AFX Head, s.d. nk nk 3p12-p21, 5p, 5q, 17p, 17q 9p, 9q, 13q, 15q

*Local recurrence. The patient with renal transplantation and immune suppression died of tumor 6 months after initial tumor diagnosis. ** Local recurrence, primary tumor not investigated. G1, Grade 1; G2, Grade 2; G3, Grade 3 Amplifications are indicated in bold. nk, not known; sc., subcutis ; s.d., superficial dermis ; s.m, skeletal muscle.

22 Mihic-Probst et al: CGH Analysis in Atypical Fibroxanthoma

Figure 2. Summary of all DNA copy number alterations detected by CGH in 24 AFX (A) and 12 UpS (B). The vertical lines on the right of the chromosome ideograms indicate gains and those on the left losses of the corresponding chromosomal regions. Bold lines represent amplifications of DNA copy number. Each line represents one alteration.

23 ANTICANCER RESEARCH 24: 19-26 (2004)

Table II. Genomic alterations in atypical fibroxanthomas (AFX) and Table III. Frequency of chromosomal alterations detected in atypical undifferentiated high grade pleomorphic sarcomas (UpS). fibroxanthomas (AFX) and undifferentiated high grade pleomorphic sarcomas (UpS). Genomic AFX UpS P alterations (n=24) (n=12) Valuea Locus of AFX UpS P alterations % (n=24) % (n=12) value* Losses 2.4±0.4b 5±0.9 0.0054 Gains 0.8±0.4 3.1±0.5 0.0014 –1q 0 25 0.0105 Amplification 0.04±0.04 0.8±0.2 0.0001 –3p 4 33 0.0171 Total 3.3±0.7 8.9±1.1 0.0001 –4q 8 25 NS –5q 0 25 0.0105 a ANOVA analysis (one factor) –6q 8 25 NS b mean±standard error –9p 54 58 NS –9q 21 0 NS –10q 4 25 NS –11p 4 33 0.0171 –11q 0 25 0.0105 –13q 42 58 NS –18q 8 25 NS –Y 25 8 NS

+4q 4 25 NS +5p 4 42 0.0044 +7q 0 25 0.0105 +8q 4 25 NS +11q 0 33 0.0027 +12q 0 25 0.0105

* Contingency table analysis NS, not significant

Figure 3. Representative examples of CGH digital images and amount and/or a larger number of MDM2 amplifications corresponding profiles illustrating amplifications of 5p and 11q in UpS (A) (26). MDM2 presumably functions as a cellular regulator or and losses of 9p and 13q in AFX (B). Tumor DNA was labeled using a mediator of the tumor suppressor protein TP53. Thus, green dUTP and normal reference DNA with red dUTP. The color ratio values 0.8, 1.2 and 1.5 were used as thresholds for chromosomal losses, increased levels of MDM2 may inactivate TP53 and thereby gains and amplifications, respectively. lead to an escape from TP53-regulated growth control (29). In UpS p53 protein inactivation is possibly due to increased levels of MDM2. By contrast, in AFX UV light- induced mutations of the p53 gene itself appear to be important (5, 6). Both events may apparently lead to the the cause of the more favorable behavior of AFX as same impairment of cell cycle regulatory mechanisms. compared to UpS. Interestingly, the 9p 21-22 region was recently linked to Previous studies suggest the amplification of 7q and 12q a rare autosomal-dominant bone dysplasia/cancer syndrome to be of particular interest for progression of UpS. One (30). This disorder is characterized by cortical growth study has shown a statistically significant correlation abnormalities and infarctions of bone, pathologic fractures between gain at 7q32 and shortened metastasis-free survival, and debilitation. Thirty-five % of the affected individuals overall survival and increased risk for local recurrence (27). also develop bone UpS. The sporadic form of this sarcoma Three genes of potential pathogenetic importance for soft accounts for 6% of all primary bone malignancies. The same tissue tumors in humans located on chromosome 12q 13-14 authors have observed identical allelic losses at chromosome have recently been identified: the sarcoma amplified 9p 21-22 in inherited and sporadic UpS (20). They sequence (SAS), the human homologue of the murine double suggested a hitherto unknown tumor suppressor gene in this minute type 2 gene (MDM2) and the cyclin dependent kinase region, because the two known genes within this region, 4 gene (CDK4) (26). Alterations of these genes have been CDKN 2A and CDKN2B, have previously been excluded as reported in UpS. Locally recurrent tumors exhibited a higher candidates by sequence analysis.

24 Mihic-Probst et al: CGH Analysis in Atypical Fibroxanthoma

Furthermore, another study suggested that the RB1 locus 10 Michie BA, Reid RP and Fallowfield ME: Aneuploidy in on chromosome 13 was lost in UpS and that biallelic atypical fibroxanthoma: DNA content quantification of 10 cases inactivation of this gene is a major, perhaps the most by image analysis. J Cutan Pathol 21(5): 404-407, 1994. important, event in the genesis of UpS (21). The high 11 el-Naggar AK, Ro JY, Ayala AG, Hinchey WW, Abdul-Karim FW and Batsakis JG: Angiomatoid malignant fibrous incidence of UpS in irradiated and in non- irradiated areas histiocytoma: flow cytometric DNA analysis of six cases. J Surg of patients previously treated for hereditary retinoblastoma Oncol 40(3): 201-204, 1989. supports this hypothesis (31). 12 Herzberg AJ, Kerns BJ, Honkanen FA, Pence JC, Iglehart JD Our results also show a large number of genetic alterations and Kinney RB: DNA ploidy and proliferation index of soft in AFX. Taken together with results of previous studies, these tissue sarcomas determined by image cytometry of fresh frozen findings confirm AFX to be a true neoplasm. Furthermore, tissue. Am J Clin Pathol 97: S29-37, 1992. our results suggest that AFX is different from UpS, though 13 Radio SJ, Wooldridge TN and Linder J: Flow cytometric DNA analysis of malignant fibrous histiocytoma and related they seem to have a common pathogenetic pathway. fibrohistiocytic tumors. Hum Pathol 19: 74-77, 1998. In addition to the confirmation of known genetic 14 Oshiro Y, Fukuda T and Tsuneyoshi M: Atypical fibroxanthoma differences between AFX and Ups, this study identified versus benign and malignant fibrous histiocytoma. A comparative genetic alterations, probably occurring later during the study of their proliferative activity using MIB-1, DNA flow course of the disease, which may serve as markers for cytometry, and p53 immunostaining. Cancer 75: 1128-1134, 1995. aggressive behavior. More detailed investigations should 15 Westermann FN, Langlois NE and Simpson JG: Apoptosis in precisely define the genes involved. atypical fibroxanthoma and pleomorphic malignant fibrous histiocytoma. Am J Dermatopathol 19: 228-231, 1997. 16 Longacre TA, Smoller BR and Rouse RV: Atypical Acknowledgements fibroxanthoma. Multiple immunohistologic profiles. Am J Surg Pathol 17: 1199-1209, 1993. We are grateful to N. Wey for photographic and computer-assisted 17 Enzinger FM and Weiss SW: Soft Tissue Tumours: Atypical reproductions and Dr. W. Jochum, Department of Pathology, Fibroxanthoma. Mosby, St. Louis 2001; 536-539. University Hospital Zürich, Switzerland, for critical reading of the 18 Lazova R, Moynes R, May D and Scott G: LN-2 (CD74): A manuscript. marker to distinguish atypical fibroxanthoma from malignant fibrous histiocytoma. Cancer 79: 2115-2124, 1997. 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