Pediatric Pathology
ISSN: 0277-0938 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/ipdp17
Nucleolar Organizer Regions in Congenital Mesoblastic Nephroma Dominique Gaillard, Raymonde Bouvier, Elise Sonsino, Liliane Boccon Gibod, Francis Jaubert, Christian Nezelof, Claudie Scheiner, Aude Lallemand & Dominique Ploton To cite this article: Dominique Gaillard, Raymonde Bouvier, Elise Sonsino, Liliane Boccon Gibod, Francis Jaubert, Christian Nezelof, Claudie Scheiner, Aude Lallemand & Dominique Ploton (1992) Nucleolar Organizer Regions in Congenital Mesoblastic Nephroma, Pediatric Pathology, 12:6, 811-821, DOI: 10.3109/15513819209024238 To link to this article: http://dx.doi.org/10.3109/15513819209024238
Published online: 09 Jul 2009.
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Date: 25 March 2016, At: 23:50
NUCLEOLAR ORGANIZER REGIONS IN CONGENITAL MESOBLASTIC NEPHROMA
Dominique Gaillard, MD
0
Laboratoire Pol Bouin, INSERM U314, C H U Reims,
C
Hbpital Edouard Heriot, Lyon, France
France
Raymonde Bouvier, M D Downloaded by [University of Sydney Library] at 23:50 25 March 2016
Elise Sonsino, MD
17
Hbpital Robert Debri, Paris, France
Liliane Boccon Gibod, MD
0
Hbpital Trousseau, Paris, France
Francis Jaubert, MD, and Christian Nezelof, M D o Hbpital Necker-Enfants Malades, Paris, France
Claudie Scheiner, M D
0
Hbpital La Timone, Marseille, France
Aude Lallemand, MD, and Dominique Ploton, PhD
0
Laboratoire Pol Bouin,
INSERM U314, C H U Reims, France
0 A review of 78 renal tumors in patients under 6 months of age revealed 35 congenital mesoblash nephromas (CMNs). Based on cellular criteria, 14 were classified as classical, 4 as partly cellular, and I 7 as cellular C M N . The mean ages were 24, 11, and 70 days, respectively. There were 13 intrarenal tumors (stage I ) but 9 classical, 3 partly cellular, and 5 cellular C M N s extended to the perirenal f a t (stage I I ) and 5 cellular tumors ruptured (stage I I I ) . I n order to assess cellular proliferative activip, silver staining of nucleolar organizer region ( A y - N O R ) proteins was performed on 19 C M N s . The number of A g - N O R dots per cell was significantly lower in classical and partly cellular C M N than in cellular CMN, whatever the stage (P C ,01). Within the cellular C M N s , the mean number of AgN O R dots was statistically higher in the single case that recurred with fatal outcome (P C .02). Counting of A g - N O R dots appears to be a reproducible method for evaluating the biologic potential d C M N s . The number of A g - N O R dots, DNA content measurements, the histological subclassification, and the presence or absence of tumor at the surgical margins may be useful features in selecting those patients who will benefit from further treatment after nephrectomy.
KEY WORDS: renal tumor, congenital mesoblastic nephroma, nucleolar organizer reyions.
Special thanks are owed to Juan-C Canellas, Noel Bonnet, and B. Visseaux-Coletto who developed the Ag-NOR method and the software, to C . Lechki for technical assistance, and to C . Juppin for typing the manuscript. Address reprint requests to Pr. Dominique Gaillard, Laboratoire Pol Bouin INSERM U 314, 45 rue Cognacq-Jay, 51092 Reims Cedex, France.
Pediatric Patholoo, 12:811-821, 1992 Copyrght 0 1992 by Hemisphere Publishing Corporation
81 1
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INTRODUCTION During the past 25 years, several investigators have focused their attention on congenital renal tumors and tumors in the first year of life, which were often grouped as congenital or infantile Wilms’ tumors (WT). In 1967 Bolande et al. delineated the typical clinical and pathological features of a benign fibromatoid tumor ( l ) , which they called the congenital mesoblastic nephroma of infancy (CMN). In 1973 Bolande showed that half of his series of presumed Wilms’ tumors in patients under 1 year of age were in reality C M N , which explained the high cure rate in this age group (2). He reviewed 48 cases and established C M N as a benign tumor cured by simple nephrectomy. However, two papers in 1973 reported large local recurrences that led to the deaths of two children (3, 4). The first case of pulmonary metastases was reported in 1980 (5) and a spectrum of potentially malignant tumors appeared. During the next few years a new subtype, named cellular or atypical C M N , was identified (6). The age of the patient was said to be an important factor, since those under the age of 3 months seemed to have an excellent prognosis regardless of cellularity or the mitotic index (7). We reviewed 78 renal tumors from patients under 6 months of age to determine the proportion of C M N and to document their outcome over a period of 34 years. Special attention was paid to the cellular C M N because of controversies regarding its nature and course. Of the various methods for evaluating the proliferative activity of growing tissue, the argyrophil technique for nucleolar organizer regions (NORs), which identifies NOR-associated proteins, has been reported to be useful in discriminating benign from malignant lesions (8-1 1). The purpose of this study was to determine whether the number of Ag-NORs per nucleus in the spindle-shaped cells of C M N provides prognostic information in addition to that given by cellularity, mitotic index, and stage.
MATERIALS AND METHOD All available cases of renal tumors in patients under 6 months of age were collected from the files of seven divisions of pediatric surgical pathology in France over a 34-year period (1957-1990). Older patients were omitted so as to have a homogeneous series, because in Europe all patients over 6 months of age receive preoperative chemotherapy. All the slides of 78 renal tumors were reviewed independently by at least two pathologists and classified as classical, partly cellular, or cellular C M N (6), different types of W T (12), rhabdoid tumor (13), clear cell sarcoma (14), or hyperplastic nephroblastomatosis (15). The size, weight, stage, and the macroscopical data were obtained from the pathology reports. Medical records from 69 patients were reviewed with emphasis on age,
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associated pathology, extension, and follow-up. The data were obtained by correspondence and from the generous collaboration of several other medical centers (Institut Gustave Roussy, Villejuif; Centre Leon Berard, Lyon). Staining of NORs was possible in only 19 of the 35 CMNs (the blocks could not be obtained for the other cases): 6 classical, 4 partly cellular, and 9 cellular CMNs including the 5 tumor ruptures with a single instance of abdominal recurrence. Representative paraffin-embedded blocks were selected. Silver staining specific for NORs was applied as previously described (16). Sections (3 pm) were dewaxed in xylene, rinsed in 100% ethanol, postfixed in 1 : 3 glacial acetic-ethanol solution, and hydrated through ethanol baths. Staining solution was prepared fresh by mixing two volumes of aqueous silver nitrate (50%) with one volume of 2 % gelatin in a 1 % formic acid solution. Tissue sections were covered with staining solution for 15 min in dark-room conditions at 20 "C . The sections were washed in several changes of distilled water, then in a 5 % thiosulfate solution, and finally in distilled water. A light counterstain with methyl green was applied before dehydration, clearing, and mounting in Eukitt. The quantification of Ag-NOR dots was achieved by using software developed in our laboratory as previously described (17). Briefly, a video camera fitted with microscope was connected to the video input of an image memory board installed on a personal computer. The image was digitized into 512 X 512 pixels. The sections were examined under an oil-immersion objective ( x 100) and the number of NORs, identified as black dots, was counted in the nuclei of at least 100 spindle-shaped tumor cells. The contour of each nucleus was drawn with the help of a graphic mouse and segmentation of NORs was performed on the basis of a gray-level threshold. The area of NORs was computed for each nucleus and was given in number of pixels or in pm2. Moreover, to allow comparison of NOR counting in different lesions, we used a reference NOR, such as a quiescent lymphocyte, in the same section. The results were expressed as Ag-NOR mass (also called number of NOR) defined as the area of NOR in one nucleus divided by the area of one reference NOR in the lymphocyte (17). The number of Ag-NOR dots was assessed in three different types of C M N and was compared. Statistical analysis was performed using Student's t-test ( P values less than .05 were considered significant).
RESULTS In our series there were 37 WTs, 35 CMNs, 4 rhabdoid tumors, 1 hyperplastic nephroblastomatosis, and no clear cell sarcoma. The mean age was 1.5 months for patients with C M N (ranging from 3 days to 6 months), 4 months for patients with W T (4 days to 6 months), and more than 4 months for rhabdoid tumor (4.6 to 6 months). There was no significant difference in frequency between W T and C M N
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during the first 6 months of life, but in the first 3 months 75% of the 40 tumors were CMNs. The main clinicopathologic features of the CMN are summarized in Table 1.
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Clinical Records The mean age at nephrectomy was lower in classical and partly cellular tumors than in cellular CMNs. Prenatal diagnosis was made in four cases; eight patients were premature. The follow-up was mentioned in 29 cases with a mean of 8.3 years ranging from 15 months to 23 years; 27 patients were alive and free of recurrence. One contralateral nephrocalcinosis was detected several days after surgery. One Solomon syndrome (epidermal nevus syndrome) was diagnosed in a patient after 6 months of age with the development of a typical linear epidermal lesion. One girl showed thalamic calcifications during the third month, raising the possibility of tuberous sclerosis. Two patients died; one death was a complication of surgery with massive gastrointestinal bleeding 4 days after nephrectomy, and the second occurred in a child who had an intraoperative rupture followed by two recurrences 5 months apart. He died 5 months later despite chemo- and radiotherapy. TABLE 1. Clinical and Pathological Features of 35 CMNs CMN
Classical
Partly cellular
Cellular
Number of cases
14 9F,5M 24 2 7 0 9R,4L, l ? 5.5 (3 to 10) I-5,II-9
4 3F, 1 M 11 2 2 2 2R,lL,l? 6 (4 to 9) I-l,11-3
17 8F,9M 70 1 8 3 8R,5L,4? 9 ( 1 to 13) I 7, I1 5, I11
14 0 No 7 3 .lo). The mean NOR area per nucleus area was of no help in recognizing the
FIGURE 2. A cellular CMN showing cells with dense nuclei, scanty cytoplasm, and mitotic figures. Hematein-phloxin-safran,
X
250.
AG-NORS IN CMN
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TABLE 2. Quantification of the Number of Ag-NOR Dots per Cell and Nucleus Area in 19 CMNs Cellular (9) ~
Classical (6)
Partly cellular (4)
No recurrence (8)
Recurrence ( 1 )
Mean number of Ag-NOR dots per cell
2.03 (1.69-2.46) SD" 0.59
2.00 (1.28-2.56) SD 0.66
2.47 (2-3.05) SD 0.79
3.57
Mean nucleus area (pm')
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~~
CMN
33
35
40
SD 1.15
46
"SD, standard deviation
different subtypes. In Table 2, the mean nucleus area seemed to increase from classical to cellular tumors and was higher in the last case; however, these results were not significant (P > .lo).
DISCUSSION Bolande recognized C M N as a common renal tumor before 6 months of age and distinct from W T (1, 2). Although the good prognosis of classical C M N is widely accepted, there is much controversy about the prognosis of cellular C M N ; several criteria have been proposed, but none is sufficient in itself. As the number of NORs has been shown to have prognostic value for several tumors, including renal carcinoma (18), lung tumors (19), benign nevi,
FIGURE 3. Ag-NOR dots in classical CMN with a mean of two dots per cell. Ag-NOR staining,
X 1000.
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FIGURE 4. High number of Ag-NOR dots in an area of cellular CMN. Ag-NOR staining,
X
1000.
and malignant melanoma (20), we decided to study the number of NORs per cell in C M N and, more particularly, to determine whether there was any difference in the number of NORs between cellular C M N with good outcome and those with recurrence. Although the study was performed in both cellular and classical areas, the number of NORs was the same in partly cellular and classical CMNs. The fact that the number of Ag-NOR dots is lower in the present study when compared to others (18-20) may be due to a number of factors. First, the proliferative index of the C M N may be low. Second, the quantification based on our software is performed only in a single focal plane and consequently does not estimate all the Ag-NOR dots contained in one nuclear section. This can be achieved when counting is done directly without image analysis. Whatever the origin of a low Ag-NOR count, the findings are statistically significant. Thus our study clearly demonstrates a statistically significant difference between classical or partly cellular C M N and cellular C M N . Moreover, in cellular C M N , the number of Ag-NOR dots was significantly higher in the case of recurrence and death. For stage I11 with tumor rupture we could not demonstrate parameters that distinguished the cellular case that recurred from the other cellular cases with good outcome. The number of patients was too small to provide definite conclusions. In a recent study, Barrantes et al. measured the DNA content in 11 CMNs by flow cytometry (21). The cells of one cellular and one partly cellular tumor
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were aneuploid, and the proliferative index could be high in typical C M N . Pettinato et al. (22) found diploid cells in one classical and one cellular C M N , but the cellular area of a partly cellular tumor was aneuploid. These results differ from the NORs quantification: the number of NORs was the same in classical and partly cellular C M N and the last subtype seems to be a simple variant of classical C M N with good prognosis. In a case of cellular C M N , Varsa et al. reported diploid-range DNA content and chromosomes with structural abnormalities (23). The number of studied cases is too small to determine whether or not DNA content predicts changes in the behavior of C M N . Age has been considered an important factor (7). In our series 13 cellular CMNs were diagnosed before 3 months; a 52-day-old boy had a local recurrence 5 months later, leading to death. Joshi et al. described a neonatal case of cellular C M N that had a local recurrence 4 months after surgery and a second massive local recurrence at 18 months of age that led to death (6). Saba and Assuncao reported a C M N at 2 months of age with postoperative recurrence and death 3 months later (24). Therefore, if age is important to prognosis, it is not absolute. Complete resection is a major criterion for prognosis (25, 26). Sandstedt et al. reported rupture or infiltration of the renal pelvis in half of the cellular CMNs at operation (27). In more than half of our series of classical CMNs, the tumor infiltrated the renal pelvis or the capsule without adverse prognosis. During the operation five tumors ruptured, and all of these were cellular CMNs. The child who died at recurrence received only one course of chemotherapy before the first recurrence. Two other ruptured cellular C M N patients are alive and well without any chemotherapy. One with hypercellularity and a very high mitotic index received chemotherapy for 6 months and is alive at 5 years. Adjuvant therapy is recommended in patients less than 3 months old with incompletely resected tumors or intraoperative rupture (27, 28). It is important to identify the relevant prognostic criteria in cases of cellular and ruptured tumors. The mitotic index is usually said to identify malignancy, but in our experience it was higher in ruptured tumors that did not recur after treatment than in the case with poor outcome. In our retrospective study the degree of necrosis was not significant. In conclusion, several features can be considered in evaluating the prognosis of cellular C M N , but the most relevant may be the occurrence of tumor rupture during surgery, the presence of tumor at the surgical margins, and the quantification of the DNA content and the NORs. For these last two studies the number of published cases is too small to draw definitive conclusions. However, preliminary results of Ag-NOR counting appear to be of value in prognostication of C M N and could be useful for selecting those patients who need further treatment to avoid recurrence of CMN.
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REFERENCES 1 . Bolande RP, Brough AJ, Izant RJ, J . Congenital mesoblastic nephroma of infancy. A report of eight cases and the relationship to Wilms’ tumor. Pediatrics 1967;40:272-8. 2. Bolande RP. Congenital mesoblastic nephroma of infancy. Perspect Pediatr Pathol 1973; 1 :227-50. 3. Joshi VV, Kay S, Milsten R , Koontz WW, McWilliams NB. Congenital mesoblastic nephroma of infancy: Report of a case with unusual clinical behavior. Am J Clin Pathol 1973;60:811-6. 4. Walker D, Richard GA. Fetal hamartoma of the kidney. Recurrence and death of patient. J Urol 1973;110:352-3. 5. Gonzales-Crussi F, Sotelo-Avila F, Kidd JM. Malignant mesenchymal nephroma of infancy: Report of a case with pulmonary metastases. Am J Surg Pathol 1980;4:185-90. 6. Joshi VV, Kasznica J, Walters T R . Atypical mesoblastic nephroma: Pathologic characterization of a potentially aggressive variant of conventional congenital mesoblastic nephroma. Arch Pathol Lab Med 1986;110:100-6. 7. Beckwith JB, Weeks DA. Congenital mesoblastic nephroma. When should we worry? (Editorial) Arch Pathol Lab Med 1986;110:98-9. 8. Crocker J, Nar P. Nucleolar organiser regions in lymphomas. J Pathol 1987;151:111-8. 9. Egan MJ, Raafat F, Crocker J , Smith K. Nucleolar organizer regions in fibrous proliferations of childhood and infantile fibrosarcoma. J Clin Pathol 1988;41:31-3. 10. Quinn C M , Wright NA. The clinical assessment of proliferation and growth in human tumours: Evaluation of methods and applications as prognostic variables. J Pathol 1990;60:93-102. 11. Derenzini M , Ploton D. Nucleolar organizer regions in cancer cells. Int Rev Exp Pathol 1991;32: 14992. 12. Beckwith JB. Wilms’ tumor and other renal tumors of childhood. A selective review from the National Wilms’ Tumor Study Pathology Center. Hum Pathol 1983;14:481-92. 13. Weeks DA, Beckwith JB, Mierav GW, Luckey OW. Rhabdoid tumor of kidney. A report of 111 cases from the National Wilms’ Tumor Study Pathology Center. Am J Surg Pathol 1989;13:439-58. 14. Marsden HB, Lawler W. Bone metastasizing renal tumour of childhood: Histopathological and clinical review of 38 cases. Virchows Arch (Pathol Anat) 1980;387:341-51. 15. Beckwith JB, Kiviat NB, Bonadio JF. Nephrogenic rests, nephroblastomatosis, and the pathogenesis of Wilms’ tumor. Pediatr Pathol 199O;lO:1-36. 16. Ploton D, Menager M , Jeannesson P, Himber G, Pigeon F, Net JJ. Improvement in the staining and in the visualization of the argyrophilic proteins of the nucleolar organizer region at the optical level. Histochem J 1986;18:5-14. 17. Ploton D, Visseaux-Coletto B, Canellas JC, et al.: Semiautomatic quantification of silver-stained nucleolar organizer regions in tissue sections and cellular smears. Anal Quant Cytol 1992;14:14-23. 18. Delahunt B, Ribas JL, Nacey J, Bethwaite PB. Nucleolar organizer regions and prognosis in renal cell carcinoma. J Pathol 1991;163:31-7. 19. Pate1 S, Himster W. Distribution and estimation of nucleolar organizer regions in various human lung turnours. Pathol Res Pract 1991;187:68-72. 20. Orrell J M , Evans AT, Grant A. A critical evaluation of Ag-NOR counting in benign naevi and malignant melanoma. J Pathol 1991;163:239-44. 21. Barrantes JC, Toyn C , Muir KR, et al. Congenital mesoblastic nephroma: Possible prognostic and management value of assessing DNA content. J Clin Pathol 1991;44:317-20. 22. Pettinato G, Manivel JC, Wick M R , Dehner LP. Classical and cellular (atypical) congenital mesoblastic nephroma: A clinicopatholigic, ultrastructural, immunohistochemical, and flow cytometric study. Hum Pathol 1989;20:682-90. 23. Varsa EW, McConnel LT, Dressler LG, Duncan M . Atypical congenital mesoblastic nephroma. Arch Pathol Lab Med 1989;113:1078-80. 24. Saba LM, Assuncao MC. Renal tumors in children under one year of age. Ann Pathol 1988;8:136-43. 25. Gormley TS,Skoog SJ, Jones RV, Maybee D. Cellular congenital mesoblastic nephroma: What are the options? J Urol 1989;142:479-83, 89. 26. Howell CG, Othersen HB, Kiviat NE, Norkool P, Beckwith JB, D’angio GJ. Therapy and outcome in
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51 children with mesoblastic nephroma: A report of the National Wilms’ Tumor Study. J Pediatr Surg 1982;17:826-31, 27. Sandstedt B, Delemarre JFM, Krul EJ, Tournade M E Mesoblastic nephroma: A study of 29 turnours from the SIOP nephroblastoma file. Histopathology 1985;9:741-50. 28. Beckwith JB. The John Lattimer Lecture Wilms’ tumor and other renal tumors of childhood: An update. J Urol 1986;136:320-4.
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Receiued February 24, 1992 Revision accepted May 25, 1992
ISSN: 0277-0938 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/ipdp17
Nucleolar Organizer Regions in Congenital Mesoblastic Nephroma Dominique Gaillard, Raymonde Bouvier, Elise Sonsino, Liliane Boccon Gibod, Francis Jaubert, Christian Nezelof, Claudie Scheiner, Aude Lallemand & Dominique Ploton To cite this article: Dominique Gaillard, Raymonde Bouvier, Elise Sonsino, Liliane Boccon Gibod, Francis Jaubert, Christian Nezelof, Claudie Scheiner, Aude Lallemand & Dominique Ploton (1992) Nucleolar Organizer Regions in Congenital Mesoblastic Nephroma, Pediatric Pathology, 12:6, 811-821, DOI: 10.3109/15513819209024238 To link to this article: http://dx.doi.org/10.3109/15513819209024238
Published online: 09 Jul 2009.
Submit your article to this journal
Article views: 1
View related articles
Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ipdp17 Download by: [University of Sydney Library]
Date: 25 March 2016, At: 23:50
NUCLEOLAR ORGANIZER REGIONS IN CONGENITAL MESOBLASTIC NEPHROMA
Dominique Gaillard, MD
0
Laboratoire Pol Bouin, INSERM U314, C H U Reims,
C
Hbpital Edouard Heriot, Lyon, France
France
Raymonde Bouvier, M D Downloaded by [University of Sydney Library] at 23:50 25 March 2016
Elise Sonsino, MD
17
Hbpital Robert Debri, Paris, France
Liliane Boccon Gibod, MD
0
Hbpital Trousseau, Paris, France
Francis Jaubert, MD, and Christian Nezelof, M D o Hbpital Necker-Enfants Malades, Paris, France
Claudie Scheiner, M D
0
Hbpital La Timone, Marseille, France
Aude Lallemand, MD, and Dominique Ploton, PhD
0
Laboratoire Pol Bouin,
INSERM U314, C H U Reims, France
0 A review of 78 renal tumors in patients under 6 months of age revealed 35 congenital mesoblash nephromas (CMNs). Based on cellular criteria, 14 were classified as classical, 4 as partly cellular, and I 7 as cellular C M N . The mean ages were 24, 11, and 70 days, respectively. There were 13 intrarenal tumors (stage I ) but 9 classical, 3 partly cellular, and 5 cellular C M N s extended to the perirenal f a t (stage I I ) and 5 cellular tumors ruptured (stage I I I ) . I n order to assess cellular proliferative activip, silver staining of nucleolar organizer region ( A y - N O R ) proteins was performed on 19 C M N s . The number of A g - N O R dots per cell was significantly lower in classical and partly cellular C M N than in cellular CMN, whatever the stage (P C ,01). Within the cellular C M N s , the mean number of AgN O R dots was statistically higher in the single case that recurred with fatal outcome (P C .02). Counting of A g - N O R dots appears to be a reproducible method for evaluating the biologic potential d C M N s . The number of A g - N O R dots, DNA content measurements, the histological subclassification, and the presence or absence of tumor at the surgical margins may be useful features in selecting those patients who will benefit from further treatment after nephrectomy.
KEY WORDS: renal tumor, congenital mesoblastic nephroma, nucleolar organizer reyions.
Special thanks are owed to Juan-C Canellas, Noel Bonnet, and B. Visseaux-Coletto who developed the Ag-NOR method and the software, to C . Lechki for technical assistance, and to C . Juppin for typing the manuscript. Address reprint requests to Pr. Dominique Gaillard, Laboratoire Pol Bouin INSERM U 314, 45 rue Cognacq-Jay, 51092 Reims Cedex, France.
Pediatric Patholoo, 12:811-821, 1992 Copyrght 0 1992 by Hemisphere Publishing Corporation
81 1
812
D. GAILLARD ET AL.
Downloaded by [University of Sydney Library] at 23:50 25 March 2016
INTRODUCTION During the past 25 years, several investigators have focused their attention on congenital renal tumors and tumors in the first year of life, which were often grouped as congenital or infantile Wilms’ tumors (WT). In 1967 Bolande et al. delineated the typical clinical and pathological features of a benign fibromatoid tumor ( l ) , which they called the congenital mesoblastic nephroma of infancy (CMN). In 1973 Bolande showed that half of his series of presumed Wilms’ tumors in patients under 1 year of age were in reality C M N , which explained the high cure rate in this age group (2). He reviewed 48 cases and established C M N as a benign tumor cured by simple nephrectomy. However, two papers in 1973 reported large local recurrences that led to the deaths of two children (3, 4). The first case of pulmonary metastases was reported in 1980 (5) and a spectrum of potentially malignant tumors appeared. During the next few years a new subtype, named cellular or atypical C M N , was identified (6). The age of the patient was said to be an important factor, since those under the age of 3 months seemed to have an excellent prognosis regardless of cellularity or the mitotic index (7). We reviewed 78 renal tumors from patients under 6 months of age to determine the proportion of C M N and to document their outcome over a period of 34 years. Special attention was paid to the cellular C M N because of controversies regarding its nature and course. Of the various methods for evaluating the proliferative activity of growing tissue, the argyrophil technique for nucleolar organizer regions (NORs), which identifies NOR-associated proteins, has been reported to be useful in discriminating benign from malignant lesions (8-1 1). The purpose of this study was to determine whether the number of Ag-NORs per nucleus in the spindle-shaped cells of C M N provides prognostic information in addition to that given by cellularity, mitotic index, and stage.
MATERIALS AND METHOD All available cases of renal tumors in patients under 6 months of age were collected from the files of seven divisions of pediatric surgical pathology in France over a 34-year period (1957-1990). Older patients were omitted so as to have a homogeneous series, because in Europe all patients over 6 months of age receive preoperative chemotherapy. All the slides of 78 renal tumors were reviewed independently by at least two pathologists and classified as classical, partly cellular, or cellular C M N (6), different types of W T (12), rhabdoid tumor (13), clear cell sarcoma (14), or hyperplastic nephroblastomatosis (15). The size, weight, stage, and the macroscopical data were obtained from the pathology reports. Medical records from 69 patients were reviewed with emphasis on age,
Downloaded by [University of Sydney Library] at 23:50 25 March 2016
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813
associated pathology, extension, and follow-up. The data were obtained by correspondence and from the generous collaboration of several other medical centers (Institut Gustave Roussy, Villejuif; Centre Leon Berard, Lyon). Staining of NORs was possible in only 19 of the 35 CMNs (the blocks could not be obtained for the other cases): 6 classical, 4 partly cellular, and 9 cellular CMNs including the 5 tumor ruptures with a single instance of abdominal recurrence. Representative paraffin-embedded blocks were selected. Silver staining specific for NORs was applied as previously described (16). Sections (3 pm) were dewaxed in xylene, rinsed in 100% ethanol, postfixed in 1 : 3 glacial acetic-ethanol solution, and hydrated through ethanol baths. Staining solution was prepared fresh by mixing two volumes of aqueous silver nitrate (50%) with one volume of 2 % gelatin in a 1 % formic acid solution. Tissue sections were covered with staining solution for 15 min in dark-room conditions at 20 "C . The sections were washed in several changes of distilled water, then in a 5 % thiosulfate solution, and finally in distilled water. A light counterstain with methyl green was applied before dehydration, clearing, and mounting in Eukitt. The quantification of Ag-NOR dots was achieved by using software developed in our laboratory as previously described (17). Briefly, a video camera fitted with microscope was connected to the video input of an image memory board installed on a personal computer. The image was digitized into 512 X 512 pixels. The sections were examined under an oil-immersion objective ( x 100) and the number of NORs, identified as black dots, was counted in the nuclei of at least 100 spindle-shaped tumor cells. The contour of each nucleus was drawn with the help of a graphic mouse and segmentation of NORs was performed on the basis of a gray-level threshold. The area of NORs was computed for each nucleus and was given in number of pixels or in pm2. Moreover, to allow comparison of NOR counting in different lesions, we used a reference NOR, such as a quiescent lymphocyte, in the same section. The results were expressed as Ag-NOR mass (also called number of NOR) defined as the area of NOR in one nucleus divided by the area of one reference NOR in the lymphocyte (17). The number of Ag-NOR dots was assessed in three different types of C M N and was compared. Statistical analysis was performed using Student's t-test ( P values less than .05 were considered significant).
RESULTS In our series there were 37 WTs, 35 CMNs, 4 rhabdoid tumors, 1 hyperplastic nephroblastomatosis, and no clear cell sarcoma. The mean age was 1.5 months for patients with C M N (ranging from 3 days to 6 months), 4 months for patients with W T (4 days to 6 months), and more than 4 months for rhabdoid tumor (4.6 to 6 months). There was no significant difference in frequency between W T and C M N
D. GAILLARD ET AL.
814
during the first 6 months of life, but in the first 3 months 75% of the 40 tumors were CMNs. The main clinicopathologic features of the CMN are summarized in Table 1.
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Clinical Records The mean age at nephrectomy was lower in classical and partly cellular tumors than in cellular CMNs. Prenatal diagnosis was made in four cases; eight patients were premature. The follow-up was mentioned in 29 cases with a mean of 8.3 years ranging from 15 months to 23 years; 27 patients were alive and free of recurrence. One contralateral nephrocalcinosis was detected several days after surgery. One Solomon syndrome (epidermal nevus syndrome) was diagnosed in a patient after 6 months of age with the development of a typical linear epidermal lesion. One girl showed thalamic calcifications during the third month, raising the possibility of tuberous sclerosis. Two patients died; one death was a complication of surgery with massive gastrointestinal bleeding 4 days after nephrectomy, and the second occurred in a child who had an intraoperative rupture followed by two recurrences 5 months apart. He died 5 months later despite chemo- and radiotherapy. TABLE 1. Clinical and Pathological Features of 35 CMNs CMN
Classical
Partly cellular
Cellular
Number of cases
14 9F,5M 24 2 7 0 9R,4L, l ? 5.5 (3 to 10) I-5,II-9
4 3F, 1 M 11 2 2 2 2R,lL,l? 6 (4 to 9) I-l,11-3
17 8F,9M 70 1 8 3 8R,5L,4? 9 ( 1 to 13) I 7, I1 5, I11
14 0 No 7 3 .lo). The mean NOR area per nucleus area was of no help in recognizing the
FIGURE 2. A cellular CMN showing cells with dense nuclei, scanty cytoplasm, and mitotic figures. Hematein-phloxin-safran,
X
250.
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TABLE 2. Quantification of the Number of Ag-NOR Dots per Cell and Nucleus Area in 19 CMNs Cellular (9) ~
Classical (6)
Partly cellular (4)
No recurrence (8)
Recurrence ( 1 )
Mean number of Ag-NOR dots per cell
2.03 (1.69-2.46) SD" 0.59
2.00 (1.28-2.56) SD 0.66
2.47 (2-3.05) SD 0.79
3.57
Mean nucleus area (pm')
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~~
CMN
33
35
40
SD 1.15
46
"SD, standard deviation
different subtypes. In Table 2, the mean nucleus area seemed to increase from classical to cellular tumors and was higher in the last case; however, these results were not significant (P > .lo).
DISCUSSION Bolande recognized C M N as a common renal tumor before 6 months of age and distinct from W T (1, 2). Although the good prognosis of classical C M N is widely accepted, there is much controversy about the prognosis of cellular C M N ; several criteria have been proposed, but none is sufficient in itself. As the number of NORs has been shown to have prognostic value for several tumors, including renal carcinoma (18), lung tumors (19), benign nevi,
FIGURE 3. Ag-NOR dots in classical CMN with a mean of two dots per cell. Ag-NOR staining,
X 1000.
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FIGURE 4. High number of Ag-NOR dots in an area of cellular CMN. Ag-NOR staining,
X
1000.
and malignant melanoma (20), we decided to study the number of NORs per cell in C M N and, more particularly, to determine whether there was any difference in the number of NORs between cellular C M N with good outcome and those with recurrence. Although the study was performed in both cellular and classical areas, the number of NORs was the same in partly cellular and classical CMNs. The fact that the number of Ag-NOR dots is lower in the present study when compared to others (18-20) may be due to a number of factors. First, the proliferative index of the C M N may be low. Second, the quantification based on our software is performed only in a single focal plane and consequently does not estimate all the Ag-NOR dots contained in one nuclear section. This can be achieved when counting is done directly without image analysis. Whatever the origin of a low Ag-NOR count, the findings are statistically significant. Thus our study clearly demonstrates a statistically significant difference between classical or partly cellular C M N and cellular C M N . Moreover, in cellular C M N , the number of Ag-NOR dots was significantly higher in the case of recurrence and death. For stage I11 with tumor rupture we could not demonstrate parameters that distinguished the cellular case that recurred from the other cellular cases with good outcome. The number of patients was too small to provide definite conclusions. In a recent study, Barrantes et al. measured the DNA content in 11 CMNs by flow cytometry (21). The cells of one cellular and one partly cellular tumor
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were aneuploid, and the proliferative index could be high in typical C M N . Pettinato et al. (22) found diploid cells in one classical and one cellular C M N , but the cellular area of a partly cellular tumor was aneuploid. These results differ from the NORs quantification: the number of NORs was the same in classical and partly cellular C M N and the last subtype seems to be a simple variant of classical C M N with good prognosis. In a case of cellular C M N , Varsa et al. reported diploid-range DNA content and chromosomes with structural abnormalities (23). The number of studied cases is too small to determine whether or not DNA content predicts changes in the behavior of C M N . Age has been considered an important factor (7). In our series 13 cellular CMNs were diagnosed before 3 months; a 52-day-old boy had a local recurrence 5 months later, leading to death. Joshi et al. described a neonatal case of cellular C M N that had a local recurrence 4 months after surgery and a second massive local recurrence at 18 months of age that led to death (6). Saba and Assuncao reported a C M N at 2 months of age with postoperative recurrence and death 3 months later (24). Therefore, if age is important to prognosis, it is not absolute. Complete resection is a major criterion for prognosis (25, 26). Sandstedt et al. reported rupture or infiltration of the renal pelvis in half of the cellular CMNs at operation (27). In more than half of our series of classical CMNs, the tumor infiltrated the renal pelvis or the capsule without adverse prognosis. During the operation five tumors ruptured, and all of these were cellular CMNs. The child who died at recurrence received only one course of chemotherapy before the first recurrence. Two other ruptured cellular C M N patients are alive and well without any chemotherapy. One with hypercellularity and a very high mitotic index received chemotherapy for 6 months and is alive at 5 years. Adjuvant therapy is recommended in patients less than 3 months old with incompletely resected tumors or intraoperative rupture (27, 28). It is important to identify the relevant prognostic criteria in cases of cellular and ruptured tumors. The mitotic index is usually said to identify malignancy, but in our experience it was higher in ruptured tumors that did not recur after treatment than in the case with poor outcome. In our retrospective study the degree of necrosis was not significant. In conclusion, several features can be considered in evaluating the prognosis of cellular C M N , but the most relevant may be the occurrence of tumor rupture during surgery, the presence of tumor at the surgical margins, and the quantification of the DNA content and the NORs. For these last two studies the number of published cases is too small to draw definitive conclusions. However, preliminary results of Ag-NOR counting appear to be of value in prognostication of C M N and could be useful for selecting those patients who need further treatment to avoid recurrence of CMN.
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51 children with mesoblastic nephroma: A report of the National Wilms’ Tumor Study. J Pediatr Surg 1982;17:826-31, 27. Sandstedt B, Delemarre JFM, Krul EJ, Tournade M E Mesoblastic nephroma: A study of 29 turnours from the SIOP nephroblastoma file. Histopathology 1985;9:741-50. 28. Beckwith JB. The John Lattimer Lecture Wilms’ tumor and other renal tumors of childhood: An update. J Urol 1986;136:320-4.
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Receiued February 24, 1992 Revision accepted May 25, 1992
