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Multiple cytogenetic aberrations in neurofibrosarcomas complicating neurofibromatosis
Multiple Cytogenetic Aberrations in Neurofibrosarcomas Complicating Neurofibromatosis Vincent M. Riccardi and Derrick W. Elder
F o u r neurofibrosarcomas, f r o m four unrelated patients with Von Recklinghausen's n e u rofibromatosis, demonstrated multiple cytogenetic aberrations characterized by loss of chromosomes and/or polysomy, as well as multiple structural abnormalities of diverse types. These observations are noteworthy for two reasons. First, the widespread and varied nature of the chromosomal changes indicate that, in at least these instances, the transformation of a benign neurofibroma to a neurofibrosarcoma may well involve a substance having an affect at multiple chromosomal sites. Second, efforts to investigate neurofibrosarcoma pathogenesis using direct analysis of DNA structure and gene activity in neurofibrosarcomas must first take into account the chromosomal distortions characterizing each tumor.
ABSTRACT:
INTRODUCTION Von Recklinghausen's neurofibromatosis (NF-I, as designated by Riccardi [1]), is a common autosomal dominant disorder characterized by multiple hyperpigmented patches of skin, hamartomatous nodules of the ocular iris, and multiple tumors of the skin and nerves, namely neurofibromas, as well as central nervous system tumors (e.g., astrocytomas), skeletal dysplasias, vascular dysplasias, functional deficits (e.g., seizures, learning disabilities, short stature) and, finally, the propensity for the neurofibromas to undergo malignant transformation [1-5]. The cancerous nerve-sheath tumors resulting from this malignant transformation are of multiple histologic types [6, 7], but the most common and characteristic type is the neurofibrosarcoma, also known as a malignant schwannoma. Although the histology and the ultrastructure of these cancers have been studied well [6, 7], there is a dearth of information about associated chromosomal anomalies or other, more subtle (i.e., molecular), genetic distortions. Moreover, other soft-tissue sarcomas are known to manifest chromosome aberrations [8], and in at least some instances the sarcoma-associated chromosome changes appear to be nonrandom [9]. In this context, we set out to examine the karyotypes of NF-associated neurofibrosarcomas for two basic reasons. First, the observed chromosome aberrations might, in their own right, provide clues about the pathogenesis of NF and the malignant transformation process. Second, because the study of these tumors by direct analysis of their DNA
From the NF Program, Baylor College of Medicine, Houston, TX.
Address r e q u e s t s for r e p r i n t s to Dr. V. M. Riccardi, NF Program, Baylar College of Medicine, 1 Baylor Plaza, H o u s t o n , TX 77030. Received September 19, 1985; accepted November 8, 1985.
199 © 1986 Elsevier Science Publishing Co., Inc. 52 Vanderbilt Ave., New York, lffY 10017
Cancer Genet Cytogenet 23:199-209 (1986) 0165-4608/86/$03.50
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v . M . Riccardi and D. W. Elder structure and genetic activity is currently underway in our own and other laboratories, the need to characterize neurofibrosarcoma chromosomes as a prelude to the molecular analyses seemed obvious.
MATERIALS
AND METHODS
Four neurofibrosarcomas from four unrelated patients with Von Recklinghausen's disease (NF-I) were available for study. Case 1
A 9-year-old black girl (NF80-172-1) born in 1971, was referred to the Baylor NF program (BNFP) for evaluation of her previously diagnosed NF-I, with specific concern for the recent development of headaches and possible seizures. Cafe-au-lait spots (CLS) and multiple cutaneous neurofibromas were present at birth. The diagnosis of NF was made in 1975 after partial excision of a large right flank plexiform neurofibroma. There was no previous family history of NF and it was presumed to represent a new mutation in her. At 3 months of age cardiac catheterization revealed pulmonary valvular and infundibular stenosis, which was treated surgically at 3 years of age. One year later, the bulk of the right flank tumor was surgically removed. At the time of our formal evaluation in 1982 there was substantial developmental delay and general incoordination. An ocular examination revealed iris Lisch nodules and amblyopia. Psychometric testing showed IQ values below 45. A cranial/ orbital CT scan and an electroencephalogram were both normal. Twenty-four hour urinary adrenalin secretion was twice the upper limit of normal, while noradrenalin secretion was within normal limits. A routine clinic visit in January 1983 revealed increasing pain over the right posterior flank, and growth of a more prominent medial portion of the previously noted plexiform neurofibroma. Blood pressure elevation to 130/98 was present. In October 1984, she developed increasing radicular back pain and the right flank tumor was noted to be enlarging. Also noted at that time were frequent, irregular menstruation, decreasing vision in the left eye, decreased range of motion in the right lower extremity, and bilaterally diminished deep tendon reflexes. She was promptly admitted to the hospital and a cranial CT scan demonstrated a 1-cm mass in the hypothalamic stalk/pituitary gland. She underwent surgical removal of the mass, which showed multifocal hyperplasia of thyrotrophs. She was readmitted in December 1984 for resection of the right flank tumor, which proved to be neurofibroma with focal malignant Triton tumor and areas of liposarcoma (i.e., mixed sarcoma including rhabdomyosarcoma, malignant Schwannoma, and liposarcoma.) In January 1985 she underwent further radical chestwall resection and liver biopsies. The resected tissue and liver biopsies were completely benign. (This patient provided specimens 1A and lB.) Case 2
A 46-year-old white woman (NF85-588-1) referred herself to the BNFP in late 1984 out of concern for a malignant Schwannoma of the lateral right ankle. CLS and cutaneous neurofibromas were noted in early childhood. A left lower extremity plexiform neurofibroma developed at age 2. NF was diagnosed at 6 years. Following numerous surgeries to the left lower extremity, apparently with resultant peroneal nerve sectioning, she had significant difficulty with ambulation. Additionally, she had remarkable facial disfigurement due to a large glabellar neurofibroma. There were no other family members known or thought to have NF. In December 1983 she noted enlargement of the lateral right ankle. Persistent
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pain and swelling led to a biopsy and partial excision of a malignant Schwannoma. Repeat surgery in June 1984 also demonstrated malignant Schwannoma. Symptoms continued to worsen, although no further treatment was instituted. In November 1984, following trauma to the right ankle, radiographs showed further growth of the tumor. An extensive evaluation failed to detect evidence of metastases, and a below-the-knee amputation was performed in January 1985. The surgical specimen showed a localized neurofibrosarcoma and surgical margins free of malignant cells. (This patient provided specimens 2A and 2B.) Case 3
A 24-year-old Latin American woman (NF82-361-1) born in 1957, was referred to us for routine evaluation of her previously diagnosed NF, with special concern for neurologic deficits that had developed over the prior 18 months. Although CLS were present at birth and neurofibromas since age 5, she was not diagnosed with NF until January 1982; she was adopted and her family history unknown. In the 18 months prior to our evaluation, she noted progressive upper and lower extremity weakness, increased temperature sensitivity in her hands, and weak hand grasps, bilaterally. On examination, she was noted to have scoliosis, pectus excavatum, areolar neurofibromas, and widespread freckling. She had a large subcutaneous neurofibroma on the left medial calf. Her neurologic examination indicated quadriparesis secondary to a massive cervical plexiform neurofibroma. A 3 x 5 cm neurofibroma of hard rubber consistency was noted on the anterior head of the right deltoid muscle. It was biopsied at the time of surgical removal of a portion of her paraspinal cervical plexiform neurofibroma. Several weeks later she required emergency surgery for cervical vertebral collapse associated with her plexiform neurofibroma, which, however, at that time demonstrated multiple areas of neurofibrosarcoma. She died approximately 6 weeks later; autopsy permission was not granted. (This patient provided specimens 3A and 3B.) Case 4
A black woman (NF83-483-1) born in 1957; was referred to us for routine evaluation of her previously diagnosed NF. She first noted neurofibromas in 1975. The diagnosis was made in 1976, at age 19 years. Two of her three children exhibited CLS, with presumed NF. None of her eight siblings or parents were thought to have NF. Her initial examination was unremarkable except for the presence of multiple neurofibromas and CLS. In October 1984, approximately 1 year after initial evaluation by us, she developed a "lump" on her left shoulder. A left forequarter amputation was performed in January 1985 for the removal of a brachial plexus neurofibrosarcoma. (This patient provided specimens 4A, 4B, and 4C.)
For each case a benign neurofibroma (from a different anatomic site) and/or a portion of skin (free of neurofibroma) was obtained, as well. In no instance was there prior radiation therapy or chemotherapy. The majority of each specimen was frozen in liquid nitrogen (for later studies), and a small segment (approximately 60 mm 3) was explanted as follows. The specimen was washed with sterile culture medium (Eagle's MEM, with Earle's salts, containing 10% fetal bovine serum, and penicillin and streptomycin) and then minced with scalpel blades. Small fragments were scratched into the surface of plastic tissue culture Petri dishes, flooded with flesh medium, and incubated at 37°C (5% CO2). After 7-21 days in culture the cells were removed from the Petri dish by treatment with trypsin (0.25%) and passaged to
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v . M . Riccardi and D. W. Eider culture slides (Lab-Tek), cultured another 36-72 hours, and then treated in situ for cytogenetic analysis. This was a c c o m p l i s h e d by treatment with colchicine for 15 minutes, followed by treatment with 0.075 M KC| for 30 minutes (at room temperature), and then four consecutive changes of ice-cold Carnoy's fixative (3:1 methanol/glacial acetic acid), and finally air-drying at room temperature. After aging at room temperature for 24-72 hours, each slide was heated to 100°C in an oven for 5 minutes, then GTG-banded by standard techniques.
RESULTS Neurofibrosarcomas
Specimen 1A. T w e n t y - n i n e m e t a p h a s e cells could have their chromosomes counted with reasonable accuracy. The stemline c h r o m o s o m e n u m b e r was basically 41 (with 2 - 5 ring chromosomes); the first side line had 81 c h r o m o s o m e s (with 4 - 6 rings), and a second side line had 155 c h r o m o s o m e s (with 9-12 rings). There were 10 cells in the first category, 14 cells in the second category, four in the third category, and one cell had a c h r o m o s o m e count of 117 (with 12 rings). Deduced karyotype signatures for the stem line and first side line were as follows (Fig. 1): 4 1 , X , - 1 , - 1 , - 2 , - 4 , - 5 , - 7 , - 8 , - 9 , - 9 , - 1 0 , - 1 1 , - 1 4 , - 1 4 , - 1 6 , - 17, +M2, + M3, +M4,+M5,+M6,+M7,+M8,+M9,+M10,+M11,+M12,(+3 rings)/77 or 81,XX, - 1 , - 1, +3, +3, - 9 , - 9 , +12, +12, +13, +13, - 1 4 , - 1 4 , +15, +15, +17, +18, +18, +21, +21, +22, +22, [_+ 2M1], +2M2, + 2 M 3 , +2M4, +2M5, +2M6, +2M7, +2M8, + 2M9, + 2M10, + 2 M l l , + 2M12,[ _+ 2M13],( + 6 rings). The second side line had 2n m u l t i p l e s of the normal and marker c h r o m o s o m e s of the first side line (and, therefore, m u l t i p l e s of the stem line, as well). M1 a p p e a r e d to be an intact c h r o m o s o m e arm l q and unidentified material (?16p) replacing l p ; a less likely interpretation was s i m p l y a l p deletion: del(1)(qter--~p22.1:). M2 a p p e a r e d to represent an asymmetric d u p l i c a t i o n of l q with an unidentified intervening sequence: 1qter-+1q12.3 : :?: : lq12.1--~1qter. M3 a p p e a r e d to be a c h r o m o s o m e # 7 with unidentified a d d i t i o n a l material attached to its distal short arm: der(7)(7qter--~7p22: :?). M4 was metacentric and of A-group size, with unidentified components. M5 was c o m p r i s e d of elements from chromosomes # 8 and #1: der(8)(8pter-*8q24: :lp33-+1pter). M6 was c o m p r i s e d of elements from c h r o m o s o m e s # 6 and #5: der(6)(6pter--~6q25: :5q31-~5qter). M7 was c o m p r i s e d of two c h r o m o s o m e s #14, with a s y m m e t r y in the pericentromeric long arm regions: der(14)(14qter--~14p12: :14q11.2--~14qter). M8 was c o m p r i s e d of one c h r o m o s o m e #14 and most of the long arm of c h r o m o s o m e #1: der(14)(14qter~ 14p11: :1q12.3--~1qter). M9 was a deleted c h r o m o s o m e #9: del(9)(qter--~p11.2:). M10 a p p e a r e d to be a d u p l i c a t i o n of 9p: dup(9)(pter--~q13: :p11-+pter). M l l , M12, M13, and the ring c h r o m o s o m e s were of unidentified origin, as were several other less consistent markers. In the stem line, for c h r o m o s o m e s #3, #12, #13, #15, and # 1 8 - 2 2 , there were two intact homologs and no a p p a r e n t a d d i t i o n a l material by virtue of structural rearrangements. For c h r o m o s o m e s # 2 , #4, #10, #11, #17, and X, only one intact homolog was present, and no a d d i t i o n a l material resulting from structural rearrangements was apparent. For c h r o m o s o m e #1, all or most of the long arm was present thrice (M2 and M9) and the distal short arm was present once (M5). In the first side line, an a d d i t i o n a l l q was present (M1). For c h r o m o s o m e #5, one normal homolog was present and distal 5q was also present (M6}. For c h r o m o s o m e #6, one normal h o m o l o g was present, and an almost c o m p l e t e homolog was contained in M6. For c h r o m o s o m e #7, one n o r m a l homolog was present, and an a p p a r e n t l y c o m p l e t e homolog was contained in M3. For c h r o m o s o m e #8, one normal homolog
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Cytogenetic Aberrations in NF
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was present, and an apparently complete homolog was contained in MS. For chromosome #9, no normal homolog was apparent, but a single representation of 9q was apparent as M9, and a double representation of 9p is apparent as M10. No normal homolog of chromosome #14 was present, but three copies of at least 14q were present (two as M7, and one as part of M8). For chromosome #16, only one intact homolog is present. In the first side line, perhaps as part of M1, 16p may have been present, as well. Other chromosomal material could not be identified at all (M4, M l l , M12, M13, rings). Specimen 2A. Each of 12 metaphase cells showed an apparently normal 46,XX chromosome pattern. One cell each with 44 and 45 counts had missing chromosomes, apparently on a random basis. Five additional cells showed the consistent presence of multiple marker chromosomes (Fig. 2). One cell (II) was characterized as follows: 4 8 , - 1 , - 7 , - 1 1 , - 1 2 , - 1 2 , - 1 3 , + 1 5 , + D , - 1 7 , - 1 7 , - 1 9 , + 2 0 , + F , + F ,
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Figure 2 Partial karyotypes showing marker chromosomes of four cells (I-IV) from specimen 2A (neurofibrosarcoma).
+G,+MI,+M2,+M3,+M4,+M4,+M7,+M8. One cell was 5 0 , X X , - 1 , - 7 , - 8 , - 1 1 , - 1 2 , - 1 2 , - 1 3 , - 1 7 , - 1 7 , +E, - 1 9 , +20, +20, +20, +F, +F, + F , +M1, +M2, + M 3 , + M 4 , + M 4 , + M 7 , + M 8 . One cell (IV) was 5 8 , X , - 1 , - 4 , - 5 , - 5 , + B , + B , - 7 , - 8 , - 9 , - 9 , - 1 2 , - 1 2 , +C, +C, +C, +C, +C, +C, +C, - 1 3 , + D , - 1 7 , +E, - 1 9 , +20, +20, + F , +F, - 2 1 , - 2 2 , +G, +G, +G, +M1, +M2, +M3, +M3, +M4, +M6, +M8. One cell (III) was 6 1 , X X , - 1 , - 1 , - 2 , - 5 , + B , + B , 7,-10,-12,- 12,+C,+C, +C, +C, - 1 3 , - 1 7 , - 1 7 , +E, +E, +E, +E, +E, - 1 9 , +F, + F , + F , +F, - 2 1 , - 2 2 , + G, + G, + M1, + M1, + M2, + M3, + M3, + M4, + M4, + M5, + M6, + M7. One cell (I) was 112,XXXX, +21, +2, +3, +3, +4, +5, +5, +6, +6, - 7 , +8, +8, +9, +9, +10, +11, +11, - 1 2 , - 1 2 , +C, +14, +14, +15, +15, +D, +D, +D, +D, - 1 7 , - 1 7 , +E, +19, +19, +20, +20, +20, +20, +20, + F , + F , + F , +F, +F, +F, + F , +F, +F, +F, +21, +21, +22, +22, +M1, +M1, +M2, +M2, +M3, +M3, +M4, +M4, +M4, +M4, + M5, + MS~ + M5, + M6, + M6, + M8, + M8. That is, as revealed in both near-diploid and near-tetraploid cells, the stem line involved m o n o s o m y 1, m o n o s o m y 7, monosomy 11, n u l l i s o m y 12, m o n o s o m y 13, m o n o s o m y 17, m o n o s o m y 19, the singular presence of M 1 - M 3 and M5-M8, the double presence of M4, and the presence of one D-group-size marker, one E-group-size marker, and multiple F-group-size markers. There was mosaicism for a normal cell line and one characterized by multiple chromosome losses and the presence of multiple marker chromosomes, the origin of which is uncertain: 46,XX/53, XX, - 1, - 7, - 12, - 12, - 13, - 17, - 19, + M1, +M2, +M3; +M3, +M4, +M4, +M5, +M6, +M7, +M8, +MD, +ME, + M F , + M F . The signature for the abnormal cell line is an extrapolation and a rough estimation, and presumes that some of the unidentified chromosomes are normal homologs, at least some of the time.
Specimen 3A. Each of six metaphase cells showed an apparent 46,XX chromosome pattern, and one cell gave a 45 count with an apparent r a n d o m loss of a chromo-
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some. Sixteen additional cells gave counts estimated as follows (multiple breaks and overlaps often precluded exact counts): one cell with 55 chromosomes, one with 57, four with 58, three with 59, four with 60, two with 61, and one with 62. Multiple cells were noted to have very large numbers of chromosome breaks, but without conspicuous structural changes, such as quadriradials. Karyotype signatures for five cells could be estimated as follows (Fig. 3). One cell (IV) was 59,XX, - 1 , +2, - 5 , - 1 2 , +D, +E, +E, +F, +F, +21, +22, +M1, +M2, +M3, +M6, +M16, +M18, +M19, +M20, +M21.Onecellwas61,XX, +A, +A, - 5 , - 6 , +C, +C, +C,-13,+18,+F,+F,+21,+21,+22,+M1,+M1,+M3,+M6,+M10 (and chromatid breaks were present thrice, in lp, 4q, and 11q). One cell (I) was 6 0 , X , - 1 , - 1 , +der(1), +2, - 5 , - 5 , - 6 , - 9 , - 1 3 , +18, +18, +20, +21, +21, - 2 2 , - 2 2 , +M1, +M2, +M3, +M4, +M5, +M6, +M6, +M7, +M8, +M10, + M l l , +M12, +M12, + M 1 3 , + M 1 4 , + M 1 5 , + M 1 6 , + M 1 7 . One cell (II) had at least 53 chromosomes; overlaps precluded an accurate count and characterization, but the following were obvious: + 2 , + 1 8 , + 1 8 , + M 1 , + M 2 , + M 6 , + M 8 , + M 1 0 , + M 1 2 , + M 1 4 , and there were two intact homologs for chromosomes #1, #3, #6, #7, #8-16, and #19-21. One cell (III) was tetraploid and showed multiple markers, including one copy of M1 and two copies of M2. Thus, the most consistent findings were trisomy 2, monosomy 5, tetrasomy 18, trisomy 20, trisomy or tetrasomy 21, and trisomy or nullisomy 22, plus the presence of M1 in all karyotypes of abnormal cells, M2 in all but one of them, and the variable presence of at least 19 other marker chromosomes.
Specimen 4A. Each of 21 metaphase cells show a basic 46,XX chromosome pattern, and nine additional cells gave counts of 51-58 (Fig. 4). One cell was as follows: 53, X, +2, - 3 , - 6 , +7, +8, - 9 , - 9 , - 1 0 , +12, +C, +C, +C, +C, +C, - 1 3 , - 1 3 , - 1 5 , +16, - 1 8 , +E, - 1 9 , - 1 9 , - 2 0 , - 2 0 , +F, +F, +2M1, +M2, +M3, +2M4,
Figure 3 Partialkaryotypes showing marker chromosomes of four cells (I-IV) from specimen 3A (neurofibrosarcoma).
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v . M . Riccardi and D. W. Elder
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Figure4 Partialkaryotypes showing marker chromosomes of three cells (I-III) from specimen 4A (neurofibrosarcoma).
+M5, +M6. One cell was 54,X, +1, +2, +5, +7, +7, + 8 , - 9 , +10, +11, - 1 3 , - 1 3 , - 1 6 , - 1 9 , - 2 0 , - 2 2 , +M1, +M2, +M3, +2M4, +M6, +M7, +Ma. One cell was 54,XX, +2, +5, +7, +7, +8, +11, - 1 3 , - 1 4 , - 1 5 , +F, +F, - 2 2 , - 2 2 , +M1, +2M2, +M4, +M5. One cell was 55, X, +2, +5, - 6 , +7, +7, +8, +12, +C, +C, +C, +C, - 1 3 , - 1 3 , - 1 5 , - 1 9 , - 2 0 , +F, - 2 2 , +M1, +M2, +M3, +2M4, +M7. One cell was 58, X, +2, - 4 , - 5 , +B, +7, +7, +C, +C, +C, +C, +C, - 1 3 , - 1 3 , - 1 4 , - 1 6 , - 1 9 , +M1, +2M2, + M 3 , + 2 M 4 . In sum, there appeared to be more or less consistent monosomy X, trisomy 2, trisomy 5, tetrasomy 7, nullisomy 13, and the consistent presence of M1-M7.
Control Cell Strains
Specimen lB. This fibroblast culture derived from a skin specimen obtained at the time the neurofibrosarcoma was removed, showed a normal 46,XX chromosome pattern (17 cells). Specimen 2B. Skin-derived fibroblasts from a specimen obtained from the right lower limb at the time of amputation showed a normal 46,XX chromosome pattern (18 cells). Specimen 3B. Fibroblasts derived from the right deltoid neurofibroma showed a normal 46,XX chromosome pattern (5 cells). Specimen 4B. Skin fibroblasts from a specimen obtained from the left arm (at the time of amputation) showed a normal 46,XX chromosome pattern (20 cells).
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Specimen 4C. A benign neurofibroma from the right anterior chest, obtained at the same time as specimens 4A and 4B, showed a normal 46,XX chromosome pattern in 33 explanted fibroblasts. Eight additional cells were chromosomally abnormal (Fig. 5): Three cells were 47,XX, + 18; (e.g., V, VI); one cell (VII) showed a proximal 2q chromatid break; one cell (II) was 46,XX,t(1;21)(q23;q11); one cell (III) was 46,XX,del(3)(pter-~q12 :); one cell (IV) was 46,XX,-19,+ ?der(7)(qter-*q14:); and one cell (I) was tetraploid, 92,XXXX,-6,-6,-7,+der(6)(6pter--*6q27::7p11--* 7pter), + del(7)(qter-*q11.1), + del(6)(pter-*q15 :), + acen fra (6)(qter-*q16). Ten additional lymphocyte, lymphoblast, skin fibroblast, and tumor cell lines cultured and processed for cytogenetic analysis during this same period failed to show the types of abnormalities detected in specimen 4C. DISCUSSION One previous report [8] documented multiple chromosome abnormalities in an NF neurofibrosarcoma. The data from that report and the neurofibrosarcoma data presented here fail to indicate any consistently abnormal chromosome or chromosomal
Figure 5 Partial karyotypes showing chromosome abnormalities seen in seven cells (I-VII) from specimen 4C (neurofibroma).
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segment. Rather, it seems that the chromosome aberrations in NF neurofibrosarcomas involve multiple sites, with both net losses and net gains of a variety of chromosome segments. That is, the key feature seems to be structural chromosome distortions at multiple sites, as opposed to consistent i n v o l v e m e n t of a specific chromosomal segment as is seen with embryonal tumors, such as Wilms' tumor [10] and retinoblastoma [11]. But, how does one account for multiple, ostensibly nonspecific sites of chromosome breaks and rearrangements a c c o m p a n y i n g (or contributing to) the development of a neurofibrosarcoma? Is there a substance (or, less satisfactorally, a phen o m e n o n ] that predisposes to chromosome breakage, which in turn predisposes to the development of the neurofibrosarcoma cellular phenotype? Although there is no direct evidence for such a p h e n o m e n o n (let alone such a substance), two additional items are noteworthy. First is the demonstration of multiple chromosome aberrations in the ostensibly benign neurofibroma cells (specimen 4C) reported above (Fig. 5). It is as though the course of this patient's NF reached a critical point wherein the effect of the NF mutation was manifest as chromosome breakage and rearrangement in neurofibromas, and eventually leading to an aberrant clonal phenotype designated a neurofibrosarcoma (i.e., specimen 4A). Second, a report by Hafez et al. [12] suggests that the NF genotype predisposes to inducible chromosome instability, consistent with the observations noted here. On the other hand, at least one other explanted NF sarcoma, an angiosarcoma, has been shown to have a normal chromosome pattern (and that tumor was the patient's third sarcoma) [3]. Finally, a note of caution. Respecting the neurofibrosarcoma chromosome abnormalities noted here, any molecular biology analysis of such tumors must consider attendant chromosomal aberrations because they may, themselves, explain distortions in the availability and transcriptional activity of a variety of specific genes.
Supported in part by the Texas Neurofibromatosis Foundation, The Neurofibromatosis Institute, and Grant CA 32064 from the NIH. The authors thank Dr. Laurens Pickard, Dr. Richard Kearns, Dr. Robert C. L. Robertson, Dr. Marvin M. Romsdahl, Dr. Thomas Wheeler, and Dr. Clayton Goodman for their assistance in obtaining the tumor and tissue specimens.
REFERENCES
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dia-Wilms' tumor association: The critical role of chromosome band 11p13. Cancer Genet Cytogenet 2:131-137. 11. Strong LC, Riccardi VM, Ferrell RE, Sparkes RS (1981): Familial retinoblastoma and chromosome 13 deletion transmitted via an insertional translocation. Science 213:1501-1503. 12. Hafez M, Sharaf L, E1-Nabi SMA, El-Wehedy G (1985): Evidence of chromosomal instability in neurofibromatosis. Cancer 55:2434-2436.
F o u r neurofibrosarcomas, f r o m four unrelated patients with Von Recklinghausen's n e u rofibromatosis, demonstrated multiple cytogenetic aberrations characterized by loss of chromosomes and/or polysomy, as well as multiple structural abnormalities of diverse types. These observations are noteworthy for two reasons. First, the widespread and varied nature of the chromosomal changes indicate that, in at least these instances, the transformation of a benign neurofibroma to a neurofibrosarcoma may well involve a substance having an affect at multiple chromosomal sites. Second, efforts to investigate neurofibrosarcoma pathogenesis using direct analysis of DNA structure and gene activity in neurofibrosarcomas must first take into account the chromosomal distortions characterizing each tumor.
ABSTRACT:
INTRODUCTION Von Recklinghausen's neurofibromatosis (NF-I, as designated by Riccardi [1]), is a common autosomal dominant disorder characterized by multiple hyperpigmented patches of skin, hamartomatous nodules of the ocular iris, and multiple tumors of the skin and nerves, namely neurofibromas, as well as central nervous system tumors (e.g., astrocytomas), skeletal dysplasias, vascular dysplasias, functional deficits (e.g., seizures, learning disabilities, short stature) and, finally, the propensity for the neurofibromas to undergo malignant transformation [1-5]. The cancerous nerve-sheath tumors resulting from this malignant transformation are of multiple histologic types [6, 7], but the most common and characteristic type is the neurofibrosarcoma, also known as a malignant schwannoma. Although the histology and the ultrastructure of these cancers have been studied well [6, 7], there is a dearth of information about associated chromosomal anomalies or other, more subtle (i.e., molecular), genetic distortions. Moreover, other soft-tissue sarcomas are known to manifest chromosome aberrations [8], and in at least some instances the sarcoma-associated chromosome changes appear to be nonrandom [9]. In this context, we set out to examine the karyotypes of NF-associated neurofibrosarcomas for two basic reasons. First, the observed chromosome aberrations might, in their own right, provide clues about the pathogenesis of NF and the malignant transformation process. Second, because the study of these tumors by direct analysis of their DNA
From the NF Program, Baylor College of Medicine, Houston, TX.
Address r e q u e s t s for r e p r i n t s to Dr. V. M. Riccardi, NF Program, Baylar College of Medicine, 1 Baylor Plaza, H o u s t o n , TX 77030. Received September 19, 1985; accepted November 8, 1985.
199 © 1986 Elsevier Science Publishing Co., Inc. 52 Vanderbilt Ave., New York, lffY 10017
Cancer Genet Cytogenet 23:199-209 (1986) 0165-4608/86/$03.50
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v . M . Riccardi and D. W. Elder structure and genetic activity is currently underway in our own and other laboratories, the need to characterize neurofibrosarcoma chromosomes as a prelude to the molecular analyses seemed obvious.
MATERIALS
AND METHODS
Four neurofibrosarcomas from four unrelated patients with Von Recklinghausen's disease (NF-I) were available for study. Case 1
A 9-year-old black girl (NF80-172-1) born in 1971, was referred to the Baylor NF program (BNFP) for evaluation of her previously diagnosed NF-I, with specific concern for the recent development of headaches and possible seizures. Cafe-au-lait spots (CLS) and multiple cutaneous neurofibromas were present at birth. The diagnosis of NF was made in 1975 after partial excision of a large right flank plexiform neurofibroma. There was no previous family history of NF and it was presumed to represent a new mutation in her. At 3 months of age cardiac catheterization revealed pulmonary valvular and infundibular stenosis, which was treated surgically at 3 years of age. One year later, the bulk of the right flank tumor was surgically removed. At the time of our formal evaluation in 1982 there was substantial developmental delay and general incoordination. An ocular examination revealed iris Lisch nodules and amblyopia. Psychometric testing showed IQ values below 45. A cranial/ orbital CT scan and an electroencephalogram were both normal. Twenty-four hour urinary adrenalin secretion was twice the upper limit of normal, while noradrenalin secretion was within normal limits. A routine clinic visit in January 1983 revealed increasing pain over the right posterior flank, and growth of a more prominent medial portion of the previously noted plexiform neurofibroma. Blood pressure elevation to 130/98 was present. In October 1984, she developed increasing radicular back pain and the right flank tumor was noted to be enlarging. Also noted at that time were frequent, irregular menstruation, decreasing vision in the left eye, decreased range of motion in the right lower extremity, and bilaterally diminished deep tendon reflexes. She was promptly admitted to the hospital and a cranial CT scan demonstrated a 1-cm mass in the hypothalamic stalk/pituitary gland. She underwent surgical removal of the mass, which showed multifocal hyperplasia of thyrotrophs. She was readmitted in December 1984 for resection of the right flank tumor, which proved to be neurofibroma with focal malignant Triton tumor and areas of liposarcoma (i.e., mixed sarcoma including rhabdomyosarcoma, malignant Schwannoma, and liposarcoma.) In January 1985 she underwent further radical chestwall resection and liver biopsies. The resected tissue and liver biopsies were completely benign. (This patient provided specimens 1A and lB.) Case 2
A 46-year-old white woman (NF85-588-1) referred herself to the BNFP in late 1984 out of concern for a malignant Schwannoma of the lateral right ankle. CLS and cutaneous neurofibromas were noted in early childhood. A left lower extremity plexiform neurofibroma developed at age 2. NF was diagnosed at 6 years. Following numerous surgeries to the left lower extremity, apparently with resultant peroneal nerve sectioning, she had significant difficulty with ambulation. Additionally, she had remarkable facial disfigurement due to a large glabellar neurofibroma. There were no other family members known or thought to have NF. In December 1983 she noted enlargement of the lateral right ankle. Persistent
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pain and swelling led to a biopsy and partial excision of a malignant Schwannoma. Repeat surgery in June 1984 also demonstrated malignant Schwannoma. Symptoms continued to worsen, although no further treatment was instituted. In November 1984, following trauma to the right ankle, radiographs showed further growth of the tumor. An extensive evaluation failed to detect evidence of metastases, and a below-the-knee amputation was performed in January 1985. The surgical specimen showed a localized neurofibrosarcoma and surgical margins free of malignant cells. (This patient provided specimens 2A and 2B.) Case 3
A 24-year-old Latin American woman (NF82-361-1) born in 1957, was referred to us for routine evaluation of her previously diagnosed NF, with special concern for neurologic deficits that had developed over the prior 18 months. Although CLS were present at birth and neurofibromas since age 5, she was not diagnosed with NF until January 1982; she was adopted and her family history unknown. In the 18 months prior to our evaluation, she noted progressive upper and lower extremity weakness, increased temperature sensitivity in her hands, and weak hand grasps, bilaterally. On examination, she was noted to have scoliosis, pectus excavatum, areolar neurofibromas, and widespread freckling. She had a large subcutaneous neurofibroma on the left medial calf. Her neurologic examination indicated quadriparesis secondary to a massive cervical plexiform neurofibroma. A 3 x 5 cm neurofibroma of hard rubber consistency was noted on the anterior head of the right deltoid muscle. It was biopsied at the time of surgical removal of a portion of her paraspinal cervical plexiform neurofibroma. Several weeks later she required emergency surgery for cervical vertebral collapse associated with her plexiform neurofibroma, which, however, at that time demonstrated multiple areas of neurofibrosarcoma. She died approximately 6 weeks later; autopsy permission was not granted. (This patient provided specimens 3A and 3B.) Case 4
A black woman (NF83-483-1) born in 1957; was referred to us for routine evaluation of her previously diagnosed NF. She first noted neurofibromas in 1975. The diagnosis was made in 1976, at age 19 years. Two of her three children exhibited CLS, with presumed NF. None of her eight siblings or parents were thought to have NF. Her initial examination was unremarkable except for the presence of multiple neurofibromas and CLS. In October 1984, approximately 1 year after initial evaluation by us, she developed a "lump" on her left shoulder. A left forequarter amputation was performed in January 1985 for the removal of a brachial plexus neurofibrosarcoma. (This patient provided specimens 4A, 4B, and 4C.)
For each case a benign neurofibroma (from a different anatomic site) and/or a portion of skin (free of neurofibroma) was obtained, as well. In no instance was there prior radiation therapy or chemotherapy. The majority of each specimen was frozen in liquid nitrogen (for later studies), and a small segment (approximately 60 mm 3) was explanted as follows. The specimen was washed with sterile culture medium (Eagle's MEM, with Earle's salts, containing 10% fetal bovine serum, and penicillin and streptomycin) and then minced with scalpel blades. Small fragments were scratched into the surface of plastic tissue culture Petri dishes, flooded with flesh medium, and incubated at 37°C (5% CO2). After 7-21 days in culture the cells were removed from the Petri dish by treatment with trypsin (0.25%) and passaged to
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v . M . Riccardi and D. W. Eider culture slides (Lab-Tek), cultured another 36-72 hours, and then treated in situ for cytogenetic analysis. This was a c c o m p l i s h e d by treatment with colchicine for 15 minutes, followed by treatment with 0.075 M KC| for 30 minutes (at room temperature), and then four consecutive changes of ice-cold Carnoy's fixative (3:1 methanol/glacial acetic acid), and finally air-drying at room temperature. After aging at room temperature for 24-72 hours, each slide was heated to 100°C in an oven for 5 minutes, then GTG-banded by standard techniques.
RESULTS Neurofibrosarcomas
Specimen 1A. T w e n t y - n i n e m e t a p h a s e cells could have their chromosomes counted with reasonable accuracy. The stemline c h r o m o s o m e n u m b e r was basically 41 (with 2 - 5 ring chromosomes); the first side line had 81 c h r o m o s o m e s (with 4 - 6 rings), and a second side line had 155 c h r o m o s o m e s (with 9-12 rings). There were 10 cells in the first category, 14 cells in the second category, four in the third category, and one cell had a c h r o m o s o m e count of 117 (with 12 rings). Deduced karyotype signatures for the stem line and first side line were as follows (Fig. 1): 4 1 , X , - 1 , - 1 , - 2 , - 4 , - 5 , - 7 , - 8 , - 9 , - 9 , - 1 0 , - 1 1 , - 1 4 , - 1 4 , - 1 6 , - 17, +M2, + M3, +M4,+M5,+M6,+M7,+M8,+M9,+M10,+M11,+M12,(+3 rings)/77 or 81,XX, - 1 , - 1, +3, +3, - 9 , - 9 , +12, +12, +13, +13, - 1 4 , - 1 4 , +15, +15, +17, +18, +18, +21, +21, +22, +22, [_+ 2M1], +2M2, + 2 M 3 , +2M4, +2M5, +2M6, +2M7, +2M8, + 2M9, + 2M10, + 2 M l l , + 2M12,[ _+ 2M13],( + 6 rings). The second side line had 2n m u l t i p l e s of the normal and marker c h r o m o s o m e s of the first side line (and, therefore, m u l t i p l e s of the stem line, as well). M1 a p p e a r e d to be an intact c h r o m o s o m e arm l q and unidentified material (?16p) replacing l p ; a less likely interpretation was s i m p l y a l p deletion: del(1)(qter--~p22.1:). M2 a p p e a r e d to represent an asymmetric d u p l i c a t i o n of l q with an unidentified intervening sequence: 1qter-+1q12.3 : :?: : lq12.1--~1qter. M3 a p p e a r e d to be a c h r o m o s o m e # 7 with unidentified a d d i t i o n a l material attached to its distal short arm: der(7)(7qter--~7p22: :?). M4 was metacentric and of A-group size, with unidentified components. M5 was c o m p r i s e d of elements from chromosomes # 8 and #1: der(8)(8pter-*8q24: :lp33-+1pter). M6 was c o m p r i s e d of elements from c h r o m o s o m e s # 6 and #5: der(6)(6pter--~6q25: :5q31-~5qter). M7 was c o m p r i s e d of two c h r o m o s o m e s #14, with a s y m m e t r y in the pericentromeric long arm regions: der(14)(14qter--~14p12: :14q11.2--~14qter). M8 was c o m p r i s e d of one c h r o m o s o m e #14 and most of the long arm of c h r o m o s o m e #1: der(14)(14qter~ 14p11: :1q12.3--~1qter). M9 was a deleted c h r o m o s o m e #9: del(9)(qter--~p11.2:). M10 a p p e a r e d to be a d u p l i c a t i o n of 9p: dup(9)(pter--~q13: :p11-+pter). M l l , M12, M13, and the ring c h r o m o s o m e s were of unidentified origin, as were several other less consistent markers. In the stem line, for c h r o m o s o m e s #3, #12, #13, #15, and # 1 8 - 2 2 , there were two intact homologs and no a p p a r e n t a d d i t i o n a l material by virtue of structural rearrangements. For c h r o m o s o m e s # 2 , #4, #10, #11, #17, and X, only one intact homolog was present, and no a d d i t i o n a l material resulting from structural rearrangements was apparent. For c h r o m o s o m e #1, all or most of the long arm was present thrice (M2 and M9) and the distal short arm was present once (M5). In the first side line, an a d d i t i o n a l l q was present (M1). For c h r o m o s o m e #5, one normal homolog was present and distal 5q was also present (M6}. For c h r o m o s o m e #6, one normal h o m o l o g was present, and an almost c o m p l e t e homolog was contained in M6. For c h r o m o s o m e #7, one n o r m a l homolog was present, and an a p p a r e n t l y c o m p l e t e homolog was contained in M3. For c h r o m o s o m e #8, one normal homolog
203
Cytogenetic Aberrations in NF
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was present, and an apparently complete homolog was contained in MS. For chromosome #9, no normal homolog was apparent, but a single representation of 9q was apparent as M9, and a double representation of 9p is apparent as M10. No normal homolog of chromosome #14 was present, but three copies of at least 14q were present (two as M7, and one as part of M8). For chromosome #16, only one intact homolog is present. In the first side line, perhaps as part of M1, 16p may have been present, as well. Other chromosomal material could not be identified at all (M4, M l l , M12, M13, rings). Specimen 2A. Each of 12 metaphase cells showed an apparently normal 46,XX chromosome pattern. One cell each with 44 and 45 counts had missing chromosomes, apparently on a random basis. Five additional cells showed the consistent presence of multiple marker chromosomes (Fig. 2). One cell (II) was characterized as follows: 4 8 , - 1 , - 7 , - 1 1 , - 1 2 , - 1 2 , - 1 3 , + 1 5 , + D , - 1 7 , - 1 7 , - 1 9 , + 2 0 , + F , + F ,
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+G,+MI,+M2,+M3,+M4,+M4,+M7,+M8. One cell was 5 0 , X X , - 1 , - 7 , - 8 , - 1 1 , - 1 2 , - 1 2 , - 1 3 , - 1 7 , - 1 7 , +E, - 1 9 , +20, +20, +20, +F, +F, + F , +M1, +M2, + M 3 , + M 4 , + M 4 , + M 7 , + M 8 . One cell (IV) was 5 8 , X , - 1 , - 4 , - 5 , - 5 , + B , + B , - 7 , - 8 , - 9 , - 9 , - 1 2 , - 1 2 , +C, +C, +C, +C, +C, +C, +C, - 1 3 , + D , - 1 7 , +E, - 1 9 , +20, +20, + F , +F, - 2 1 , - 2 2 , +G, +G, +G, +M1, +M2, +M3, +M3, +M4, +M6, +M8. One cell (III) was 6 1 , X X , - 1 , - 1 , - 2 , - 5 , + B , + B , 7,-10,-12,- 12,+C,+C, +C, +C, - 1 3 , - 1 7 , - 1 7 , +E, +E, +E, +E, +E, - 1 9 , +F, + F , + F , +F, - 2 1 , - 2 2 , + G, + G, + M1, + M1, + M2, + M3, + M3, + M4, + M4, + M5, + M6, + M7. One cell (I) was 112,XXXX, +21, +2, +3, +3, +4, +5, +5, +6, +6, - 7 , +8, +8, +9, +9, +10, +11, +11, - 1 2 , - 1 2 , +C, +14, +14, +15, +15, +D, +D, +D, +D, - 1 7 , - 1 7 , +E, +19, +19, +20, +20, +20, +20, +20, + F , + F , + F , +F, +F, +F, + F , +F, +F, +F, +21, +21, +22, +22, +M1, +M1, +M2, +M2, +M3, +M3, +M4, +M4, +M4, +M4, + M5, + MS~ + M5, + M6, + M6, + M8, + M8. That is, as revealed in both near-diploid and near-tetraploid cells, the stem line involved m o n o s o m y 1, m o n o s o m y 7, monosomy 11, n u l l i s o m y 12, m o n o s o m y 13, m o n o s o m y 17, m o n o s o m y 19, the singular presence of M 1 - M 3 and M5-M8, the double presence of M4, and the presence of one D-group-size marker, one E-group-size marker, and multiple F-group-size markers. There was mosaicism for a normal cell line and one characterized by multiple chromosome losses and the presence of multiple marker chromosomes, the origin of which is uncertain: 46,XX/53, XX, - 1, - 7, - 12, - 12, - 13, - 17, - 19, + M1, +M2, +M3; +M3, +M4, +M4, +M5, +M6, +M7, +M8, +MD, +ME, + M F , + M F . The signature for the abnormal cell line is an extrapolation and a rough estimation, and presumes that some of the unidentified chromosomes are normal homologs, at least some of the time.
Specimen 3A. Each of six metaphase cells showed an apparent 46,XX chromosome pattern, and one cell gave a 45 count with an apparent r a n d o m loss of a chromo-
Cytogenetic Aberrations in NF
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some. Sixteen additional cells gave counts estimated as follows (multiple breaks and overlaps often precluded exact counts): one cell with 55 chromosomes, one with 57, four with 58, three with 59, four with 60, two with 61, and one with 62. Multiple cells were noted to have very large numbers of chromosome breaks, but without conspicuous structural changes, such as quadriradials. Karyotype signatures for five cells could be estimated as follows (Fig. 3). One cell (IV) was 59,XX, - 1 , +2, - 5 , - 1 2 , +D, +E, +E, +F, +F, +21, +22, +M1, +M2, +M3, +M6, +M16, +M18, +M19, +M20, +M21.Onecellwas61,XX, +A, +A, - 5 , - 6 , +C, +C, +C,-13,+18,+F,+F,+21,+21,+22,+M1,+M1,+M3,+M6,+M10 (and chromatid breaks were present thrice, in lp, 4q, and 11q). One cell (I) was 6 0 , X , - 1 , - 1 , +der(1), +2, - 5 , - 5 , - 6 , - 9 , - 1 3 , +18, +18, +20, +21, +21, - 2 2 , - 2 2 , +M1, +M2, +M3, +M4, +M5, +M6, +M6, +M7, +M8, +M10, + M l l , +M12, +M12, + M 1 3 , + M 1 4 , + M 1 5 , + M 1 6 , + M 1 7 . One cell (II) had at least 53 chromosomes; overlaps precluded an accurate count and characterization, but the following were obvious: + 2 , + 1 8 , + 1 8 , + M 1 , + M 2 , + M 6 , + M 8 , + M 1 0 , + M 1 2 , + M 1 4 , and there were two intact homologs for chromosomes #1, #3, #6, #7, #8-16, and #19-21. One cell (III) was tetraploid and showed multiple markers, including one copy of M1 and two copies of M2. Thus, the most consistent findings were trisomy 2, monosomy 5, tetrasomy 18, trisomy 20, trisomy or tetrasomy 21, and trisomy or nullisomy 22, plus the presence of M1 in all karyotypes of abnormal cells, M2 in all but one of them, and the variable presence of at least 19 other marker chromosomes.
Specimen 4A. Each of 21 metaphase cells show a basic 46,XX chromosome pattern, and nine additional cells gave counts of 51-58 (Fig. 4). One cell was as follows: 53, X, +2, - 3 , - 6 , +7, +8, - 9 , - 9 , - 1 0 , +12, +C, +C, +C, +C, +C, - 1 3 , - 1 3 , - 1 5 , +16, - 1 8 , +E, - 1 9 , - 1 9 , - 2 0 , - 2 0 , +F, +F, +2M1, +M2, +M3, +2M4,
Figure 3 Partialkaryotypes showing marker chromosomes of four cells (I-IV) from specimen 3A (neurofibrosarcoma).
MI
M2
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v . M . Riccardi and D. W. Elder
M2 MI
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Figure4 Partialkaryotypes showing marker chromosomes of three cells (I-III) from specimen 4A (neurofibrosarcoma).
+M5, +M6. One cell was 54,X, +1, +2, +5, +7, +7, + 8 , - 9 , +10, +11, - 1 3 , - 1 3 , - 1 6 , - 1 9 , - 2 0 , - 2 2 , +M1, +M2, +M3, +2M4, +M6, +M7, +Ma. One cell was 54,XX, +2, +5, +7, +7, +8, +11, - 1 3 , - 1 4 , - 1 5 , +F, +F, - 2 2 , - 2 2 , +M1, +2M2, +M4, +M5. One cell was 55, X, +2, +5, - 6 , +7, +7, +8, +12, +C, +C, +C, +C, - 1 3 , - 1 3 , - 1 5 , - 1 9 , - 2 0 , +F, - 2 2 , +M1, +M2, +M3, +2M4, +M7. One cell was 58, X, +2, - 4 , - 5 , +B, +7, +7, +C, +C, +C, +C, +C, - 1 3 , - 1 3 , - 1 4 , - 1 6 , - 1 9 , +M1, +2M2, + M 3 , + 2 M 4 . In sum, there appeared to be more or less consistent monosomy X, trisomy 2, trisomy 5, tetrasomy 7, nullisomy 13, and the consistent presence of M1-M7.
Control Cell Strains
Specimen lB. This fibroblast culture derived from a skin specimen obtained at the time the neurofibrosarcoma was removed, showed a normal 46,XX chromosome pattern (17 cells). Specimen 2B. Skin-derived fibroblasts from a specimen obtained from the right lower limb at the time of amputation showed a normal 46,XX chromosome pattern (18 cells). Specimen 3B. Fibroblasts derived from the right deltoid neurofibroma showed a normal 46,XX chromosome pattern (5 cells). Specimen 4B. Skin fibroblasts from a specimen obtained from the left arm (at the time of amputation) showed a normal 46,XX chromosome pattern (20 cells).
Cytogenetic Aberrations in NF
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Specimen 4C. A benign neurofibroma from the right anterior chest, obtained at the same time as specimens 4A and 4B, showed a normal 46,XX chromosome pattern in 33 explanted fibroblasts. Eight additional cells were chromosomally abnormal (Fig. 5): Three cells were 47,XX, + 18; (e.g., V, VI); one cell (VII) showed a proximal 2q chromatid break; one cell (II) was 46,XX,t(1;21)(q23;q11); one cell (III) was 46,XX,del(3)(pter-~q12 :); one cell (IV) was 46,XX,-19,+ ?der(7)(qter-*q14:); and one cell (I) was tetraploid, 92,XXXX,-6,-6,-7,+der(6)(6pter--*6q27::7p11--* 7pter), + del(7)(qter-*q11.1), + del(6)(pter-*q15 :), + acen fra (6)(qter-*q16). Ten additional lymphocyte, lymphoblast, skin fibroblast, and tumor cell lines cultured and processed for cytogenetic analysis during this same period failed to show the types of abnormalities detected in specimen 4C. DISCUSSION One previous report [8] documented multiple chromosome abnormalities in an NF neurofibrosarcoma. The data from that report and the neurofibrosarcoma data presented here fail to indicate any consistently abnormal chromosome or chromosomal
Figure 5 Partial karyotypes showing chromosome abnormalities seen in seven cells (I-VII) from specimen 4C (neurofibroma).
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v . M . Riccardi and D. W. Elder
segment. Rather, it seems that the chromosome aberrations in NF neurofibrosarcomas involve multiple sites, with both net losses and net gains of a variety of chromosome segments. That is, the key feature seems to be structural chromosome distortions at multiple sites, as opposed to consistent i n v o l v e m e n t of a specific chromosomal segment as is seen with embryonal tumors, such as Wilms' tumor [10] and retinoblastoma [11]. But, how does one account for multiple, ostensibly nonspecific sites of chromosome breaks and rearrangements a c c o m p a n y i n g (or contributing to) the development of a neurofibrosarcoma? Is there a substance (or, less satisfactorally, a phen o m e n o n ] that predisposes to chromosome breakage, which in turn predisposes to the development of the neurofibrosarcoma cellular phenotype? Although there is no direct evidence for such a p h e n o m e n o n (let alone such a substance), two additional items are noteworthy. First is the demonstration of multiple chromosome aberrations in the ostensibly benign neurofibroma cells (specimen 4C) reported above (Fig. 5). It is as though the course of this patient's NF reached a critical point wherein the effect of the NF mutation was manifest as chromosome breakage and rearrangement in neurofibromas, and eventually leading to an aberrant clonal phenotype designated a neurofibrosarcoma (i.e., specimen 4A). Second, a report by Hafez et al. [12] suggests that the NF genotype predisposes to inducible chromosome instability, consistent with the observations noted here. On the other hand, at least one other explanted NF sarcoma, an angiosarcoma, has been shown to have a normal chromosome pattern (and that tumor was the patient's third sarcoma) [3]. Finally, a note of caution. Respecting the neurofibrosarcoma chromosome abnormalities noted here, any molecular biology analysis of such tumors must consider attendant chromosomal aberrations because they may, themselves, explain distortions in the availability and transcriptional activity of a variety of specific genes.
Supported in part by the Texas Neurofibromatosis Foundation, The Neurofibromatosis Institute, and Grant CA 32064 from the NIH. The authors thank Dr. Laurens Pickard, Dr. Richard Kearns, Dr. Robert C. L. Robertson, Dr. Marvin M. Romsdahl, Dr. Thomas Wheeler, and Dr. Clayton Goodman for their assistance in obtaining the tumor and tissue specimens.
REFERENCES
1. Riccardi VM (1982): Neurofibromatosis: Clinical heterogeneity. Curr Prob Cancer 7:1-35. 2. Riccardi VM (1981): Von Recklinghausen neurofibromatosis. N Engl J Med 305:1617-1627. 3. Riccardi VM, Wheeler TM, Pickard LR, King B (1984): The pathophysiology of neurofibromatosis. II. Angiosarcoma as a complication. Cancer Genet Cytogenet 12:275-280. 4. Hope DG, Mulvihill JJ (1981): Malignancy in neurofibromatosis. Adv Neurol 29:33-56. 5. Sorensen SV, Mulvihill JJ, Nielsen A (1986): 35-year follow-up of neurofibromatosis. Ann NY Acad Sci (in press). 6. Guccion JG, Enzinger FM (1979): Malignant Schwannoma associated with von Recklinghausen neurofibromatosis. Virch Arch (Pathol Anat) 383:43-57. 7. Herrea GA, Pinto de Moraes H (1984): Neurogenic sarcomas in patients with neurofibromatosis (von Recklinghausen's disease). Virch Arch (Pathol Anat) 403:361-376. 8. Becher R, Wake N, Gibas Z, Sandberg AA (1984): Chromosome changes in soft tissue sarcomas. J Natl Cancer Inst 72:823-831. 9. Trent J, Casper J, Meltzer P, Thomphson F, Fogh J (1985): Nonrandom chromosome alterations in rhabdomyosarcoma. Cancer Genet Cytogenet 16:189-197. 10. Riccardi VM, Hittner HM, Francke U, Yunis JJ, Ledbetter D, Borges W (1980): The aniri-
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