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Presence or absence of trisomy 11 is correlated with histologic subtype in congenital mesoblastic nephroma
Presence or Absence of Trisomy 11 is Correlated With Histotogic Subtype in r L-d M -hi tlr_ Np.nhr J. T. Mascarello, T. R. Cajulis, H. F. Krous, and P. M. Carpenter
ABSTRACT: Fluorescence in situ hybridization utilizing a probe for the alpha satellite repeat sequence on chromosome 11 was used to detect variations in the number of chromosomes 11 in 24 formalin-fixed, paraffin-embedded congenital mesoblastic nephromas. Evidence of trisomy 11 was found in nearly half of the tumors. More importantly, the presence of trisomy 11 was associated with the cellular histologic variant of this tumor.
INTRODUCTION Congenital mesoblastic nephroma (CMN) is a rare tumor occurring in the kidneys of neonates [1-3]. The histopathology of CMN has prompted past investigators to designate it as a fetal mesenchymal hamartoma [4], a leiomyomatous hamartoma [5], and a congenital fibromatoma [6]. In 1967, Bolande et al. [1] distinguished CMN from Wilms' tumor on the basis of features common to eight cases previously identified as monotypic spindle cell Wilms' tumors. Histologically these tumors resembled leiomyoma, a benign tumor of smooth muscle origin found most commonly in the uterus or muscularis of the gastrointestinal tract. Subsequently a more cellular variant characterized by high cellularity, numerous mitoses, and occasional foci of hemorrhage or necrosis was recognized [2, 3, 7, 8]. Tumors in which cellular elements occur against an otherwise classic background have been included in the cellular category. CMN is usually cured by complete surgical excision but metastasis and recurrence have been reported in cases with cellular histology [3, 9]. Five of the seven CMNs studied thus far with traditional cytogenetic methods have had karyotypes with three copies of chromosome 11 [10-15]. Unfortunately, CMN is so uncommon that confirmation of this apparent trend or exploration of differences between histologic subtypes would be difficult with traditional methods. Recently, fluorescence in situ hybridization (FISH] has emerged as an alternative method for studying the number of target chromosomes in formalinfixed paraffin-embedded materials (see for example [16-19]). This method directly detects labeled DNA probes that have been hybridized to complementary DNA sequences in the
From the Genetic Services (J. T. M., T. R. C.) and Pathology Department (I4. E K.), Children's Hospital-San Diego, San Diego, California; and Department of Pathology (P. M. C.), University of California at Irvine Medical Center, Orange, California. Address reprint requests to: J. T. Moscarello, Genetic Services, Children's Hospital-San Diego, 3020 Children's Way, San Diego, CA 92123. Received July 26, 1993; accepted March 2, 1994. 50 Cancer Genet Cytogenet77:50-54(1994) 0165-4608/94/$07.00
tissue being studied. By selecting the appropriate chromosome-specific probe, one can visualize the number of copies (the fluorescent signals] of that chromosome in each nucleus or section of a nucleus. While this method does not provide the cytogenetic detail of traditional methods, it does allow one to answer specific questions about targeted chromosomes in archival materials that may have been collected in many locations over a large number of years. We have used FISH to demonstrate that trisomy 11 is a common feature in 30 paraffin-embedded, archival CMN specimens and that the distribution of trisomy 11 is correlated with the cellular histologic pattern.
MATERIALS AND M E T H O D S Blocks or sections from paraffin-embedded CMNs were obtained from: Children's Hospital of Boston (13 cases]; The National Wilms' Tumor Study Pathology Center (four cases); Children's Hospital of Philadelphia (three cases]; Children's Hospital of Pittsburgh (seven cases); and Children's Hospital of San Diego (two cases, one of which had a recurrence and was, therefore, represented by two sets of sections). One of the San Diego cases had been studied previously (and reported] with traditional cytogenetic methods and was known to have trisomy 11 [among other karyotypic anomalies) [12]. Fluorescence in situ hybridization was performed on 4-~m sections using alpha satellite probes and kits for formalinfixed tissues (Oncor Inc). After pretreatment in sodium bisulfite and pepsin solutions, the slides were dehydrated and overlayed with a 2 x SSC and formamide solution containing a biotinylated DNA probe complementary to the alpha satellite sequences in the pericentromeric region of the target chromosome. Once covered and sealed with a glass coverslip, the slides were denatured for 12 minutes at 90°C and then incubated overnight at 37°C. Excess probe was washed away by repeated washings in 50% formamide in 2 x SSC (at 37°C). The signal from the hybridized probe was generated with FITC-avidin followed by a biotinylated anti-avidin
© 1994Elsevier ScienceInc.
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T r i s o m y 11 in C o n g e n i t a l M e s o b l a s t i c N e p h r o m a
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a n t i b o d y a m p l i f i c a t i o n system. N u c l e i w e r e c o u n t e r s t a i n e d w i t h 0.5 m c g / m l p r o p i d i u m iodide. In t h o s e cases exhibiting w e a k FITC f l u o r e s c e n c e after s t a n d a r d p r o c e s s i n g , the h y b r i d i z a t i o n t i m e s w e r e e x t e n d e d (up to 5 days) a n d two r o u n d s of signal a m p l i f i c a t i o n w e r e u s e d . All cases w e r e p r o b e d w i t h the c h r o m o s o m e 11-specific probe. To d e t e r m i n e t h e n u m b e r of c o p i e s of c h r o m o s o m e 11 in the sections, we c o u n t e d t h e n u m b e r of n u c l e i w i t h 1, 2, or 3 f l u o r e s c e n t signals after a p p l y i n g t h e c h r o m o s o m e 11 probe. W h e n t h e n u m b e r of n u c l e i w i t h t h r e e signals exc e e d e d 5% of the total s c o r e d (or, as in o n e case, the n u m b e r of n u c l e i w i t h o n e signal was m u c h h i g h e r t h a n t h e n u m b e r w i t h two), this r e s u l t was c o m p a r e d to the r e s u l t o b t a i n e d w i t h e i t h e r a c h r o m o s o m e 18 or a c h r o m o s o m e 12 probe. Direct d e t e r m i n a t i o n of c h r o m o s o m e c o p y n u m b e r is not possible in s e c t i o n e d tissues b e c a u s e sections do not c o n t a i n w h o l e n u c l e i a n d b e c a u s e it is n e c e s s a r y to control for scoring errors i n t r o d u c e d by one's i n a b i l i t y to r e l i a b l y i d e n t i f y t h e m a r g i n s of a d j a c e n t n u c l e i . T h e c h r o m o s o m e 18 probe was initially u s e d as the control probe (two copies expected), but b e c a u s e it p r o d u c e d a m u c h m o r e intense signal t h a n the c h r o m o s o m e 11 probe, we b e c a m e c o n c e r n e d that the difference in signal intensity m i g h t skew the results and later u t i l i z e d a c h r o m o s o m e 12 p r o b e for this p u r p o s e . In all b u t t h r e e cases {numbers 2, 20, a n d 21), it was p o s s i b l e to use a d j a c e n t s e c t i o n s for t h e test a n d control experiments. C M N is w e l l k n o w n for interdigitating w i t h or e n t r a p p i n g areas of n o r m a l kidney. By p e r f o r m i n g the F I S H analysis o n cells w i t h i n t h e i r natural architecture, we w e r e able to avoid areas w i t h n o r m a l k i d n e y a r c h i t e c t u r e and, thereby, m i n i m i z e t h e n u m e r i c a l i m p a c t of c o n t a m i n a t i n g n o r m a l cells. For all but t h e three cases in w h i c h w e exhausted our supp l y of sections, o n e of t h e sections a d j a c e n t to the section s t u d i e d w i t h F I S H was stained w i t h h e m a t o x y l i n a n d e o s i n a n d b l i n d l y c a t e g o r i z e d by two of us (P. M. C. a n d H. F. K.}. Sections r e s e m b l i n g l e i o m y o m a w i t h loosely arranged spindle cells, m o d e r a t e cellularity, a n d few m i t o s e s w e r e designated as h a v i n g t h e classic h i s t o l o g y [1]. S e c t i o n s e x h i b i t i n g h i g h c e l l u l a r i t y {either focally or diffusely} as w e l l as freq u e n t mitotic figures a n d areas of necrosis or h e m o r r h a g e w e r e c l a s s i f i e d as c e l l u l a r [2, 3, 8]. W h e r e a d j a c e n t sections w e r e not available, h i s t o l o g i c s u b t y p e was e s t a b l i s h e d f r o m a n o t h e r r e g i o n (of t h e s a m e tumor}.
ies of c h r o m o s o m e 11 t h a n any o t h e r set of control values. W h i l e c o m p a r i s o n s b e t w e e n sets of sections may be u n d e s i r able, this set of values should, nevertheless, constitute a conservative set on w h i c h to base c o m p a r i s o n s . E v i d e n c e for the p r e s e n c e of t h r e e c o p i e s of the p e r i c e n t r o m e r i c r e g i o n of c h r o m o s o m e 11 was f o u n d in n i n e cases {Tables 1 a n d 2). C o m p a r i s o n of the d i s t r i b u t i o n of signals in control e x p e r i m e n t s y i e l d e d p v a l u e s for c h i - s q u a r e analysis of less t h a n 0.005 for all but one of the nine. Surprisingly, one case a p p e a r e d to have o n l y one c o p y of t h e target region.
RESULTS
15 16
FISH was successful o n 24 of the 30 sets of sections (29 cases, o n e of w h i c h h a d a r e c u r r e n c e that was also sectioned). In m o s t cases, large n u m b e r s of i n f o r m a t i v e n u c l e i w e r e available a n d at least 150 w e r e scored. In two cases, however, diff i c u l t y in a c h i e v i n g a d e q u a t e uptake of t h e p r o b e as w e l l as t h e p r e s e n c e of an o b s c u r i n g r e s i d u e l i m i t e d t h e scoring to fewer t h a n 150 n u c l e i . In t h r e e cases, t h e s e s a m e factors c a u s e d us to use all of t h e available sections for o p t i m i z i n g the c h r o m o s o m e 11 p r o b e e x p e r i m e n t s and left n o n e for control study. We u s e d the c h r o m o s o m e 18 control v a l u e s from case 11 for c o m p a r i s o n w i t h t h e s e cases. T h e case 11 control was c h o s e n because the distribution of scores for n u c l e i w i t h one, two, or t h r e e signals m o r e closely a p p r o x i m a t e d the dist r i b u t i o n of s c o r e s in cases i n t e r p r e t e d as h a v i n g t h r e e c o p -
Table 1
F I S H signals in C M N s e c t i o n s h a v i n g classical h i s t o l o g y
Case no.
Cells with n signals
Copies of
Probe Q n = 1 n = 2 n = 3 probe target
1 2 3 4 5 6
11 11 11 11 11 11 12 11 11 11 11 11
7 8 9 10 11
92 55 63 58 64 133 94 61 72 24 71 114
96 131 90 107 84 11 63 76 90 34 101 122
4 15 7 1 4 0 2 1 4 0 4 5
2 3 2 2 2 1 2 2 2 2 2 2
;~2 p < 0.05
p < 0.005
a Signal scores for control probes are provided only for specimens in which the number of copies of chromosome 11 appeared to deviate from 2 and for which adequate numbers of sections were available.
Table 2
F I S H signals in C M N s e c t i o n s h a v i n g cellular histology
Case no. 12 13 14
17 18 19 20 21 22 23 23rec
Cells with n signals
Copies of
Probe ° n = 1 n = 2 n = 3 probe target 11 12 11 18 11 12 11 11 12 11 12 11 11 12 11 11 11 18 11 11
45 82 47 75 12 82 101 9 84 19 82 52 27 54 10 11 30 8 85 89
92 64 74 104 105 90 53 141 75 69 90 130 71 121 20 40 80 129 85 125
55 0 30 12 26 3 1 104 0 86 3 6 9 7 11 42 122 9 3 16
3 2 3 2 3 2 2 3 2 3 2 2 2 2 3 3 3 2 2 2
~2 p < 0.005 p < 0.005 p < 0.005
p < 0.005 p < 0.005
NS p < 0.005 p < 0.005 p < 0.005
a Signal scores for control probes are provided only for specimens in which the number of copies of chromosome 11 appeared to be greater than 2 and the supply of sections was adequate for control study.
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J . T . Mascarello et al.
W h e n the cases are d i v i d e d into a group with classic histology (Table 1) a n d one w i t h cellular histology (Table 2), a clear pattern emerges. Only one o f t e n cases w i t h the classic pattern (Fig. 1) showed evidence of more t h a n 2 chromosomes 11 per nucleus. C o m p a r i s o n of probe signals in that case to the case 11 control y i e l d e d a p value between 0.05 and 0.01. However, the proportion of nuclei w i t h three signals was far less than that noted in other cases judged to have three chromosomes 11 so we are m u c h less confident that cells in this t u m o r really d i d have trisomy c h r o m o s o m e 11. The case that exhibits evidence of m o n o s o m y 11 was also one with classic histology. Thirteen cases were classified as having the cellular histologic pattern (Fig. 2). Eight showed evidence of three copies of chromosome 11. Included among those w i t h cellular histology is a case that recurred (case 23). Both the original t u m o r and the recurrence had cellular histology but neither exhibited evidence of trisomy 11. Two other tumors included in this category had mixed histologic patterns. Because of the b l i n d e d nature of the study, we were not able to limit the FISH analysis to only areas of cellular histology. Nevertheless, trisomy 11 was apparent in one. DISCUSSION Traditional cytogenetic studies have been reported on seven cases of CMN [10-15]. Five h a d karyotypes with one or more chromosomes 11. The karyotype of another [11] d i d not have three copies of chromosome 11 but did contain a marker chromosome whose origin could not be identified, so the possibility that it too had extra chromatin from c h r o m o s o m e 11 cannot be excluded. The present study confirms that trisomy 11 is a c o m m o n anomaly in CM_N and, additionally, demonstrates a distinct association between trisomy 11 and the cellular histologic pattern for this tumor. Even including the one classic histology case with marginal evidence of trisomy 11, Fishers Exact Test performed on the n u m b e r of trisomy 11 versus nontrisomy cases in the two histologic types indicates a significant difference [p = 0.029) between types. Furthermore, it should be p o i n t e d out here that FISH analysis may well underestimate the frequency of karyotypic anomalies involving chromosome 11 in these tumors. Over and beyond the technical factors that might contribute to such an underestimate, one must recognize that the probe detects only the pericentromeric region of chromosome 11 and that any structural anomalies resulting in d u p l i c a t i o n of other parts of the chromosome w o u l d not be detectable. Structural anomalies have been described in cases studied w i t h traditional methods [11, 12] so this may not be a trivial matter. With respect to the association of karyotype and histologic type, the case reported by Kovacs et al. [10] is of particular interest, In that case, a tumor with mixed histology was studied with traditional cytogenetic methods. Areas of the tumor with classic histology had a normal karyotype while cellular areas h a d a h y p e r d i p l o i d karyotype that i n c l u d e d an extra copy of chromosome 11. Karyotypic data on c h i l d h o o d renal tumors other than Wilms' t u m o r are not plentiful but some observations are, nevertheless, worth n o t l n g Constitutional deletions invotv-
Figure 1 Classic histologic subtype of congenital mesoblastic nephroma, a) Low-magnification view illustrating fascicles of spindle cells entrapping non-neoplastic renal tubules, low cellularity, and absence of necrosis (hematoxylin and eosin stain, ×125). b) Higher magnification view illustrating absence of mitoses and pleomorphism. Collagen fibrils separate the individual cells {hematoxylin and eosin stain, x 312).
ing the short arm of chromosome 11 p r o d u c e a well-characterizod predisposition to Wflms' tumor and structural anomalies involving chromosome 11 reportedly occur in about 20% of all Wilms' tumors. Nevertheless, trisomy 11 is u n c o m m o n in Wflms' tumor [16, 20]. Timmons et al. [21] reported trisemy 8, 12, 17, 19, and 20 in a case of cystic partially differentiated nephroblastoma (CPDN) and, by drawing attention to the case of a boy with constitutional trisomy 8 mosaicism and a CPDN
Trisomy 11 in Congenital Mesoblastic N e p h r o m a
53 CMNs with mixed histologies [8, 10] suggest a sequential relationship between the classic and cellular variants. It is unlikely that an abnormal karyotype w o u l d revert to a normal karyotype. Consequently, CMN most likely begins as a clone of cells w i t h properties similar to (or the same as) those of the classic variant. The accumulation of trisomy 11 and (possibly) other genetic "hits" in one or more cells w o u l d then lead to a subclone or subclones with more aggressive growth properties. Because of the specific involvement of chromosome 11, one could speculate that the insulin-like growth factor II (IGFII) gene, a gene that has been m a p p e d to the short arm of c h r o m o s o m e 11 [24] and that is expressed in mesenchymal cells during the course of kidney development [25], may play a key role in the acquisition of these properties. We express our gratitude to the following individuals who either provided or helped us locate specimens for this study: Bruce Beckwith (National Wilms Tumor Study Pathology Center); Paul Dickman (Children's Hospital of Pittsburgh); Jane Chattan (Children's Hospital of Philadelphia); Harry Kozakewich (Children's Hospital of Boston). We express our appreciation for the technical assistance of Phyllis Dudley.
REFERENCES
Figure 2 Cellular subtype of congenital mesoblastic nephroma. a) Low-magnification view illustrating high cellularity, paucity of stromal collagen, and focal necrosis (left). An entrapped renal tubule is present in the upper right (hematoxylin and eosin stain, × 125). b) Higher magnification view illustrating marked pleomorphism and mitotic figures (hematoxylin and eosin stain, × 312). that was not s t u d i e d cytogenetically [22], raise the possibility that trisomy 8 might be the significant a n o m a l y in that renal neoplasm. A histologic pattern similar to that of the cellular variant of CMN is also typical of congenital fibrosarcoma so it is interesting to note that trisomy 11 has also been demonstrated in this t u m o r [23]. Finally w h e n c o n s i d e r e d together, the association of trisomy 11 with the cellular histology and the existence of
1. Bolande RP, Brough AJ, Izant RH (1967): Congenital mesoblastic nephroma of infancy. A report of eight cases and the relationship to Wilms' tumor. Pediatrics 40:272-278. 2. Chan HS, Cang M-Y,Mancer K, Payton D, Weitzman SS, Kotecha P, Daneman A (1987): Congenital mesoblastic nephroma: a clinicoradiologic study of 17 cases representing the pathologic spectrum of the disease. J Pediatr 111:64-70. 3. Gonzalez-Crussi F, Sotelo-Avila C, Kidd JM (1980): Malignant mesenchymal nephroma of infancy: report of a case with pulmonary metastases. Am J Surg Path 4:185-190. 4. Wigger HJ (1975): Fetal mesenchymal hamartoma of kidney. A tumor of secondary mesenchyme. Cancer 36:1002-1008. 5. Bogdan R, Taylor DEM, Mostofi FK (1973): Leiomyomatous hamartoma of the kidney. A clinical and pathological analysis of 20 cases from the kidney tumor registry. Cancer 31:462-467. 6. Teng P, Warder MJ, Cohn WL (1963): Congenital generalized fibromatosis (renal and skeletal) with complete spontaneous regression. J Pediatr 62:748-753. 7. Joshi W , Kasznica J, Walters TR (1986): Atypical mesoblastic nephroma: pathologic characterization of a potentially aggressive variant of conventional congenital mesoblastic nephroma. Arch Pathol Lab Med 110:100-106. 8. Pettinato G, Manivel C, Wick MR, Dehner LP (1989): Classical and cellular (atypical) congenital mesoblastic nephroma: a clinicopathologic, ultrastructural, immunohistochemical and flow cytometric study. Hum Pathol 20:682-690. 9. Fu Y-S, Kay S (1973): Congenital mesoblastic nephroma and its recurrence. Arch Pathol 96:66-70. 10. KovacsG, Szucs S, Maschek H (1987): Two chromosomally different cell populations in a partly cellular congenital mesoblastic nephroma. Arch Pathol Lab Med 111:383-385. 11. Varsa EW, McConnell TS, Dressier LG, Duncan M (1989): Atypical congenital mesoblastic nephroma: report of a case with karyotypic and flow cytometric analysis. Arch Pathol Lab Med 113:1078-1080. 12. Carpenter PM, Mascarello JT, Kaplan GW, Krous HF: Congenital mesoblastic nephroma: cytogenetic comparison to leiomyoma. Pediatr Pathol (in press). 13. Teyssier JR, Ferre D (1989): Frequent clonal chromosomal
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14. Kaneko Y, Homma C, Maseki N, Sakuri M, Hata J (1991): Correlation of chromosome abnormalities with histological and clinical features in Wilms' and other childhood renal tumors. Cancer Res 51:5937-5942. 15. Tomlinson GE, Argyle JC, Velasco S, Nessen PL (1992): Molecular characterization of congenital mesoblastic nephroma and its distinction from Wilms' Tumor. Cancer 70:2358-2361. 16. Drut RM, Harris CP, Drut R, Meisner L (1992): Use of fluorescent in situ hybridization to detect trisomy 13 in archival tissues for cytogenetic diagnosis. Pediatr Pathol 12:799-805. 17. Lutz S, Welter C, Zang KD, Seitz G, Blin N, Urbschat K (19921: A two color technique for chromosome in situ hybridization in tissue sections. J Pathol 167:279-282. 18. Mascarello JT, Cajulis TR, Billman GE, Spruce WE (1994): Ovarian germ cell tumor evolving to myelodysplasia. Genes Chrom Cancer.
19. Schofield DE, Fletcher JA (1992): Trisomy 12 in pediatric granulosa-stromal cell tumors. Am J Pathol 141:1265-1269. 20. Solis V, Pritchard J, Cowell JK (1988): Cytogenetic changes in Wilms' tumors. Cancer Genet Cytogenet 34:223-234. 21. Timmons CF, McGavran L, Untirkircher L, Beckwith JB, Wilson HL (1989): Hyperdiploidy including trisomy 8 in a cystic partially differentiated nephroblastoma. Cancer Genet Cytogenet 41:79-85. 22. Niss R, Passarge E (1976): Trisomy 8 restricted to cultured fibroblasts. J Med Genet 13:229-234. 23. Argyle JC, Tomlinson GE, Stewart 13, Schneider NR (1992): U1trastructural, immunocytochemical and cytogenetic characterization of a large congenital fibrosarcoma. Arch Pathol Lab Med 116:972-975. 24. Tricoli JV, Rail BL, Scott J, Bell GI, Shows TB (1984): Localization of insulinlike growth factor genes to human chromosomes 11 and 12. Nature 310:784-786. 25. Yun K (1993): Clear cell sarcoma of the kidney expresses insulinlike growth factor-II but not WT1 transcripts. Am J Pathol 142: 39- 47.
ABSTRACT: Fluorescence in situ hybridization utilizing a probe for the alpha satellite repeat sequence on chromosome 11 was used to detect variations in the number of chromosomes 11 in 24 formalin-fixed, paraffin-embedded congenital mesoblastic nephromas. Evidence of trisomy 11 was found in nearly half of the tumors. More importantly, the presence of trisomy 11 was associated with the cellular histologic variant of this tumor.
INTRODUCTION Congenital mesoblastic nephroma (CMN) is a rare tumor occurring in the kidneys of neonates [1-3]. The histopathology of CMN has prompted past investigators to designate it as a fetal mesenchymal hamartoma [4], a leiomyomatous hamartoma [5], and a congenital fibromatoma [6]. In 1967, Bolande et al. [1] distinguished CMN from Wilms' tumor on the basis of features common to eight cases previously identified as monotypic spindle cell Wilms' tumors. Histologically these tumors resembled leiomyoma, a benign tumor of smooth muscle origin found most commonly in the uterus or muscularis of the gastrointestinal tract. Subsequently a more cellular variant characterized by high cellularity, numerous mitoses, and occasional foci of hemorrhage or necrosis was recognized [2, 3, 7, 8]. Tumors in which cellular elements occur against an otherwise classic background have been included in the cellular category. CMN is usually cured by complete surgical excision but metastasis and recurrence have been reported in cases with cellular histology [3, 9]. Five of the seven CMNs studied thus far with traditional cytogenetic methods have had karyotypes with three copies of chromosome 11 [10-15]. Unfortunately, CMN is so uncommon that confirmation of this apparent trend or exploration of differences between histologic subtypes would be difficult with traditional methods. Recently, fluorescence in situ hybridization (FISH] has emerged as an alternative method for studying the number of target chromosomes in formalinfixed paraffin-embedded materials (see for example [16-19]). This method directly detects labeled DNA probes that have been hybridized to complementary DNA sequences in the
From the Genetic Services (J. T. M., T. R. C.) and Pathology Department (I4. E K.), Children's Hospital-San Diego, San Diego, California; and Department of Pathology (P. M. C.), University of California at Irvine Medical Center, Orange, California. Address reprint requests to: J. T. Moscarello, Genetic Services, Children's Hospital-San Diego, 3020 Children's Way, San Diego, CA 92123. Received July 26, 1993; accepted March 2, 1994. 50 Cancer Genet Cytogenet77:50-54(1994) 0165-4608/94/$07.00
tissue being studied. By selecting the appropriate chromosome-specific probe, one can visualize the number of copies (the fluorescent signals] of that chromosome in each nucleus or section of a nucleus. While this method does not provide the cytogenetic detail of traditional methods, it does allow one to answer specific questions about targeted chromosomes in archival materials that may have been collected in many locations over a large number of years. We have used FISH to demonstrate that trisomy 11 is a common feature in 30 paraffin-embedded, archival CMN specimens and that the distribution of trisomy 11 is correlated with the cellular histologic pattern.
MATERIALS AND M E T H O D S Blocks or sections from paraffin-embedded CMNs were obtained from: Children's Hospital of Boston (13 cases]; The National Wilms' Tumor Study Pathology Center (four cases); Children's Hospital of Philadelphia (three cases]; Children's Hospital of Pittsburgh (seven cases); and Children's Hospital of San Diego (two cases, one of which had a recurrence and was, therefore, represented by two sets of sections). One of the San Diego cases had been studied previously (and reported] with traditional cytogenetic methods and was known to have trisomy 11 [among other karyotypic anomalies) [12]. Fluorescence in situ hybridization was performed on 4-~m sections using alpha satellite probes and kits for formalinfixed tissues (Oncor Inc). After pretreatment in sodium bisulfite and pepsin solutions, the slides were dehydrated and overlayed with a 2 x SSC and formamide solution containing a biotinylated DNA probe complementary to the alpha satellite sequences in the pericentromeric region of the target chromosome. Once covered and sealed with a glass coverslip, the slides were denatured for 12 minutes at 90°C and then incubated overnight at 37°C. Excess probe was washed away by repeated washings in 50% formamide in 2 x SSC (at 37°C). The signal from the hybridized probe was generated with FITC-avidin followed by a biotinylated anti-avidin
© 1994Elsevier ScienceInc.
655 Avenueof the Americas,New York, NY 10010
T r i s o m y 11 in C o n g e n i t a l M e s o b l a s t i c N e p h r o m a
51
a n t i b o d y a m p l i f i c a t i o n system. N u c l e i w e r e c o u n t e r s t a i n e d w i t h 0.5 m c g / m l p r o p i d i u m iodide. In t h o s e cases exhibiting w e a k FITC f l u o r e s c e n c e after s t a n d a r d p r o c e s s i n g , the h y b r i d i z a t i o n t i m e s w e r e e x t e n d e d (up to 5 days) a n d two r o u n d s of signal a m p l i f i c a t i o n w e r e u s e d . All cases w e r e p r o b e d w i t h the c h r o m o s o m e 11-specific probe. To d e t e r m i n e t h e n u m b e r of c o p i e s of c h r o m o s o m e 11 in the sections, we c o u n t e d t h e n u m b e r of n u c l e i w i t h 1, 2, or 3 f l u o r e s c e n t signals after a p p l y i n g t h e c h r o m o s o m e 11 probe. W h e n t h e n u m b e r of n u c l e i w i t h t h r e e signals exc e e d e d 5% of the total s c o r e d (or, as in o n e case, the n u m b e r of n u c l e i w i t h o n e signal was m u c h h i g h e r t h a n t h e n u m b e r w i t h two), this r e s u l t was c o m p a r e d to the r e s u l t o b t a i n e d w i t h e i t h e r a c h r o m o s o m e 18 or a c h r o m o s o m e 12 probe. Direct d e t e r m i n a t i o n of c h r o m o s o m e c o p y n u m b e r is not possible in s e c t i o n e d tissues b e c a u s e sections do not c o n t a i n w h o l e n u c l e i a n d b e c a u s e it is n e c e s s a r y to control for scoring errors i n t r o d u c e d by one's i n a b i l i t y to r e l i a b l y i d e n t i f y t h e m a r g i n s of a d j a c e n t n u c l e i . T h e c h r o m o s o m e 18 probe was initially u s e d as the control probe (two copies expected), but b e c a u s e it p r o d u c e d a m u c h m o r e intense signal t h a n the c h r o m o s o m e 11 probe, we b e c a m e c o n c e r n e d that the difference in signal intensity m i g h t skew the results and later u t i l i z e d a c h r o m o s o m e 12 p r o b e for this p u r p o s e . In all b u t t h r e e cases {numbers 2, 20, a n d 21), it was p o s s i b l e to use a d j a c e n t s e c t i o n s for t h e test a n d control experiments. C M N is w e l l k n o w n for interdigitating w i t h or e n t r a p p i n g areas of n o r m a l kidney. By p e r f o r m i n g the F I S H analysis o n cells w i t h i n t h e i r natural architecture, we w e r e able to avoid areas w i t h n o r m a l k i d n e y a r c h i t e c t u r e and, thereby, m i n i m i z e t h e n u m e r i c a l i m p a c t of c o n t a m i n a t i n g n o r m a l cells. For all but t h e three cases in w h i c h w e exhausted our supp l y of sections, o n e of t h e sections a d j a c e n t to the section s t u d i e d w i t h F I S H was stained w i t h h e m a t o x y l i n a n d e o s i n a n d b l i n d l y c a t e g o r i z e d by two of us (P. M. C. a n d H. F. K.}. Sections r e s e m b l i n g l e i o m y o m a w i t h loosely arranged spindle cells, m o d e r a t e cellularity, a n d few m i t o s e s w e r e designated as h a v i n g t h e classic h i s t o l o g y [1]. S e c t i o n s e x h i b i t i n g h i g h c e l l u l a r i t y {either focally or diffusely} as w e l l as freq u e n t mitotic figures a n d areas of necrosis or h e m o r r h a g e w e r e c l a s s i f i e d as c e l l u l a r [2, 3, 8]. W h e r e a d j a c e n t sections w e r e not available, h i s t o l o g i c s u b t y p e was e s t a b l i s h e d f r o m a n o t h e r r e g i o n (of t h e s a m e tumor}.
ies of c h r o m o s o m e 11 t h a n any o t h e r set of control values. W h i l e c o m p a r i s o n s b e t w e e n sets of sections may be u n d e s i r able, this set of values should, nevertheless, constitute a conservative set on w h i c h to base c o m p a r i s o n s . E v i d e n c e for the p r e s e n c e of t h r e e c o p i e s of the p e r i c e n t r o m e r i c r e g i o n of c h r o m o s o m e 11 was f o u n d in n i n e cases {Tables 1 a n d 2). C o m p a r i s o n of the d i s t r i b u t i o n of signals in control e x p e r i m e n t s y i e l d e d p v a l u e s for c h i - s q u a r e analysis of less t h a n 0.005 for all but one of the nine. Surprisingly, one case a p p e a r e d to have o n l y one c o p y of t h e target region.
RESULTS
15 16
FISH was successful o n 24 of the 30 sets of sections (29 cases, o n e of w h i c h h a d a r e c u r r e n c e that was also sectioned). In m o s t cases, large n u m b e r s of i n f o r m a t i v e n u c l e i w e r e available a n d at least 150 w e r e scored. In two cases, however, diff i c u l t y in a c h i e v i n g a d e q u a t e uptake of t h e p r o b e as w e l l as t h e p r e s e n c e of an o b s c u r i n g r e s i d u e l i m i t e d t h e scoring to fewer t h a n 150 n u c l e i . In t h r e e cases, t h e s e s a m e factors c a u s e d us to use all of t h e available sections for o p t i m i z i n g the c h r o m o s o m e 11 p r o b e e x p e r i m e n t s and left n o n e for control study. We u s e d the c h r o m o s o m e 18 control v a l u e s from case 11 for c o m p a r i s o n w i t h t h e s e cases. T h e case 11 control was c h o s e n because the distribution of scores for n u c l e i w i t h one, two, or t h r e e signals m o r e closely a p p r o x i m a t e d the dist r i b u t i o n of s c o r e s in cases i n t e r p r e t e d as h a v i n g t h r e e c o p -
Table 1
F I S H signals in C M N s e c t i o n s h a v i n g classical h i s t o l o g y
Case no.
Cells with n signals
Copies of
Probe Q n = 1 n = 2 n = 3 probe target
1 2 3 4 5 6
11 11 11 11 11 11 12 11 11 11 11 11
7 8 9 10 11
92 55 63 58 64 133 94 61 72 24 71 114
96 131 90 107 84 11 63 76 90 34 101 122
4 15 7 1 4 0 2 1 4 0 4 5
2 3 2 2 2 1 2 2 2 2 2 2
;~2 p < 0.05
p < 0.005
a Signal scores for control probes are provided only for specimens in which the number of copies of chromosome 11 appeared to deviate from 2 and for which adequate numbers of sections were available.
Table 2
F I S H signals in C M N s e c t i o n s h a v i n g cellular histology
Case no. 12 13 14
17 18 19 20 21 22 23 23rec
Cells with n signals
Copies of
Probe ° n = 1 n = 2 n = 3 probe target 11 12 11 18 11 12 11 11 12 11 12 11 11 12 11 11 11 18 11 11
45 82 47 75 12 82 101 9 84 19 82 52 27 54 10 11 30 8 85 89
92 64 74 104 105 90 53 141 75 69 90 130 71 121 20 40 80 129 85 125
55 0 30 12 26 3 1 104 0 86 3 6 9 7 11 42 122 9 3 16
3 2 3 2 3 2 2 3 2 3 2 2 2 2 3 3 3 2 2 2
~2 p < 0.005 p < 0.005 p < 0.005
p < 0.005 p < 0.005
NS p < 0.005 p < 0.005 p < 0.005
a Signal scores for control probes are provided only for specimens in which the number of copies of chromosome 11 appeared to be greater than 2 and the supply of sections was adequate for control study.
52
J . T . Mascarello et al.
W h e n the cases are d i v i d e d into a group with classic histology (Table 1) a n d one w i t h cellular histology (Table 2), a clear pattern emerges. Only one o f t e n cases w i t h the classic pattern (Fig. 1) showed evidence of more t h a n 2 chromosomes 11 per nucleus. C o m p a r i s o n of probe signals in that case to the case 11 control y i e l d e d a p value between 0.05 and 0.01. However, the proportion of nuclei w i t h three signals was far less than that noted in other cases judged to have three chromosomes 11 so we are m u c h less confident that cells in this t u m o r really d i d have trisomy c h r o m o s o m e 11. The case that exhibits evidence of m o n o s o m y 11 was also one with classic histology. Thirteen cases were classified as having the cellular histologic pattern (Fig. 2). Eight showed evidence of three copies of chromosome 11. Included among those w i t h cellular histology is a case that recurred (case 23). Both the original t u m o r and the recurrence had cellular histology but neither exhibited evidence of trisomy 11. Two other tumors included in this category had mixed histologic patterns. Because of the b l i n d e d nature of the study, we were not able to limit the FISH analysis to only areas of cellular histology. Nevertheless, trisomy 11 was apparent in one. DISCUSSION Traditional cytogenetic studies have been reported on seven cases of CMN [10-15]. Five h a d karyotypes with one or more chromosomes 11. The karyotype of another [11] d i d not have three copies of chromosome 11 but did contain a marker chromosome whose origin could not be identified, so the possibility that it too had extra chromatin from c h r o m o s o m e 11 cannot be excluded. The present study confirms that trisomy 11 is a c o m m o n anomaly in CM_N and, additionally, demonstrates a distinct association between trisomy 11 and the cellular histologic pattern for this tumor. Even including the one classic histology case with marginal evidence of trisomy 11, Fishers Exact Test performed on the n u m b e r of trisomy 11 versus nontrisomy cases in the two histologic types indicates a significant difference [p = 0.029) between types. Furthermore, it should be p o i n t e d out here that FISH analysis may well underestimate the frequency of karyotypic anomalies involving chromosome 11 in these tumors. Over and beyond the technical factors that might contribute to such an underestimate, one must recognize that the probe detects only the pericentromeric region of chromosome 11 and that any structural anomalies resulting in d u p l i c a t i o n of other parts of the chromosome w o u l d not be detectable. Structural anomalies have been described in cases studied w i t h traditional methods [11, 12] so this may not be a trivial matter. With respect to the association of karyotype and histologic type, the case reported by Kovacs et al. [10] is of particular interest, In that case, a tumor with mixed histology was studied with traditional cytogenetic methods. Areas of the tumor with classic histology had a normal karyotype while cellular areas h a d a h y p e r d i p l o i d karyotype that i n c l u d e d an extra copy of chromosome 11. Karyotypic data on c h i l d h o o d renal tumors other than Wilms' t u m o r are not plentiful but some observations are, nevertheless, worth n o t l n g Constitutional deletions invotv-
Figure 1 Classic histologic subtype of congenital mesoblastic nephroma, a) Low-magnification view illustrating fascicles of spindle cells entrapping non-neoplastic renal tubules, low cellularity, and absence of necrosis (hematoxylin and eosin stain, ×125). b) Higher magnification view illustrating absence of mitoses and pleomorphism. Collagen fibrils separate the individual cells {hematoxylin and eosin stain, x 312).
ing the short arm of chromosome 11 p r o d u c e a well-characterizod predisposition to Wflms' tumor and structural anomalies involving chromosome 11 reportedly occur in about 20% of all Wilms' tumors. Nevertheless, trisomy 11 is u n c o m m o n in Wflms' tumor [16, 20]. Timmons et al. [21] reported trisemy 8, 12, 17, 19, and 20 in a case of cystic partially differentiated nephroblastoma (CPDN) and, by drawing attention to the case of a boy with constitutional trisomy 8 mosaicism and a CPDN
Trisomy 11 in Congenital Mesoblastic N e p h r o m a
53 CMNs with mixed histologies [8, 10] suggest a sequential relationship between the classic and cellular variants. It is unlikely that an abnormal karyotype w o u l d revert to a normal karyotype. Consequently, CMN most likely begins as a clone of cells w i t h properties similar to (or the same as) those of the classic variant. The accumulation of trisomy 11 and (possibly) other genetic "hits" in one or more cells w o u l d then lead to a subclone or subclones with more aggressive growth properties. Because of the specific involvement of chromosome 11, one could speculate that the insulin-like growth factor II (IGFII) gene, a gene that has been m a p p e d to the short arm of c h r o m o s o m e 11 [24] and that is expressed in mesenchymal cells during the course of kidney development [25], may play a key role in the acquisition of these properties. We express our gratitude to the following individuals who either provided or helped us locate specimens for this study: Bruce Beckwith (National Wilms Tumor Study Pathology Center); Paul Dickman (Children's Hospital of Pittsburgh); Jane Chattan (Children's Hospital of Philadelphia); Harry Kozakewich (Children's Hospital of Boston). We express our appreciation for the technical assistance of Phyllis Dudley.
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Figure 2 Cellular subtype of congenital mesoblastic nephroma. a) Low-magnification view illustrating high cellularity, paucity of stromal collagen, and focal necrosis (left). An entrapped renal tubule is present in the upper right (hematoxylin and eosin stain, × 125). b) Higher magnification view illustrating marked pleomorphism and mitotic figures (hematoxylin and eosin stain, × 312). that was not s t u d i e d cytogenetically [22], raise the possibility that trisomy 8 might be the significant a n o m a l y in that renal neoplasm. A histologic pattern similar to that of the cellular variant of CMN is also typical of congenital fibrosarcoma so it is interesting to note that trisomy 11 has also been demonstrated in this t u m o r [23]. Finally w h e n c o n s i d e r e d together, the association of trisomy 11 with the cellular histology and the existence of
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