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Increased microvascular density predicts relapse in Wilms' tumor
Increased Microvascular Density Predicts Relapse in Wilms’ Tumor By Lisa P. Abramson, Paul E. Grundy, Alfred W. Rademaker, Irene Helenowski, Mona Cornwell, Howard M. Katzenstein, Marleta Reynolds, Robert M. Arensman, and Susan E. Crawford Chicago, Illinois and Edmonton, Alberta
Background/Purpose: Tumor stage and histology are the most important prognostic criteria in Wilms’ tumors; however, a subset of patients remains who have favorable histology tumors and unexpectedly relapse. The authors postulated that increased microvascular density (MVD), a hallmark for angiogenesis, could identify patients at risk for relapse.
evaluating the favorable histology (FH) group alone, there was higher MVD in the relapse group (32.4 ⫹/⫺ 2.7 v 19 ⫹/⫺ 1.8; P ⬍ .05). MVD was found to be the only predictor of relapse when compared with age, sex, tumor weight, and histology.
Methods: A case-control study was used to compare relapse (n ⫽ 15) with nonrelapse tumors (n ⫽ 35). Tumor MVD was counted in 5 random high-powered fields (hpf) using antiFactor VIII antibody and expressed as mean vessel count/hpf ⫹/⫺ SEM. MVD and clinical data were evaluated using univariate analysis and student’s t test.
Conclusions: These results suggest that increased MVD can identify Wilms’ tumor patients at high risk for relapse, especially those patients with favorable histology tumors. A larger study is warranted to determine the potential utility of MVD in stratification of Wilms’ tumor patients. J Pediatr Surg 38:325-330. Copyright 2003, Elsevier Science (USA). All rights reserved.
Results: The relapse group had higher MVD than the nonrelapse group (34.9 ⫹/⫺ 2.9 v 22.4 ⫹/⫺ 2; P ⬍ .05). When
INDEX WORDS: Wilms’ tumor, microvascular density, angiogenesis, favorable histology, relapse.
W
ILMS’ TUMOR (WT) is the second most common solid abdominal tumor in childhood with overall survival rates exceeding 90%. Despite significant therapeutic advances, there still remains a subset of patients with favorable prognostic indicators who unexpectedly relapse and do not respond to aggressive second line therapy.1 This patient population continues to be the source of intensive investigation by The National Wilms’ Tumor Study Group (NWTSG), which currently is conducting its fifth study. In addition to stage, a variety of clinical and tumor-related factors have been evaluated as potential markers for poor outcome. Multivariate analysis has shown that blastemal WT suppressor gene (WT-1) expression is an independent prognostic marker for disease progression,2 whereas other studies have linked poor outcome to changes in the expression of the tumor suppressor gene p533-5 and/or loss of heterozygosity on chromosomes 16q, 1q, and 22q.6-9 The spectrum of genetic alterations in WT led to the incorporation of a prospective genetic screen in NWTSG-V. It is too early in the investigation to know if any one of these genetic indices will help identify patients at risk for recurrent disease. Despite the vast amount of genetic data accumulated from WT tissue, standard gross and histologic evaluation continues to provide important prognostic information. During NWTSG-1, anaplastic histology was found to be one of the most powerful predictors of relapse,10 and those patients with diffuse anaplasia consistently did not respond to an intensified therapeutic regimen.11 Recog-
nizing that the majority of relapsed Wilms’ tumors have favorable histology (FH), other factors clearly contribute to the aggressive phenotype. To date, one of the most difficult tasks for the oncologists has been identifying the group of patients with lower-stage FH tumors who are at risk for recurrence. When this specific subset of patients has been studied, increased p53,12 gain of 1q,13 and high telomerase reverse transcriptase (hTERT)14 were shown to positively correlate with relapse suggesting that one or more of these may prove to be useful markers. Knowing that angiogenesis is required for tumors to grow, invade, and metastasize15,16 and that pathologic angiogenesis is involved in the progression of Wilms’ tumor (WT),17 we chose to examine the predictive value of tissue microvascular density (MVD), a hallmark of angiogenesis. MVD was an important prognostic indica-
Journal of Pediatric Surgery, Vol 38, No 3 (March), 2003: pp 325-330
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From the Divisions of Pediatric Surgery and Hematology-Oncology, Children’s Memorial Hospital, Departments of Biostatistics and Pathology, Northwestern University Medical School, Chicago, IL, and the Department of Pediatrics, Cross Cancer Institute, Edmonton, Alberta, Canada. Presented at the 33rd Annual Meeting of the American Pediatric Surgical Association, Phoenix, Arizona, May 19-23, 2002. Supported by Grant CA64239 from the National Cancer Institute. Address reprint requests to Susan E. Crawford, MD, Department of Pathology, W-127, Northwestern University Medical School, 303 E Chicago Ave, Chicago, IL 60611. Copyright 2003, Elsevier Science (USA). All rights reserved. 0022-3468/03/3803-0010$35.00/0 doi:10.1053/jpsu.2003.50102
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tor in a wide variety of carcinomas including breast, prostate, and non–small cell lung.18 This pilot study sought to determine if higher MVD correlated with more aggressive biological behavior in Wilms’ tumor patients. Furthermore, we evaluated the subgroup of patients with FH to determine if MVD may be able to identify those children without adverse prognostic features who relapsed. If MVD had predictive value, this would allow earlier intensive treatment and heightened surveillance in patients with highly vascular tumors. MATERIALS AND METHODS
Sample Selection and Tissue Acquisition A case-control study was used to compare biological differences in tumors from patients with and without relapsed Wilms’ tumors. A review of all WT patients treated at a single institution from 1981 through 2001 (n ⫽ 88) was undertaken to identify relapse patients (n ⫽ 12). Three additional cases of relapsed patients were obtained from the NWTSG tissue bank. Nonrelapsed controls were obtained from both the NWTSG and the Children’s Memorial Hospital with a minimum disease-free follow-up of 2 years (n ⫽ 35). Patients were staged clinically and treated according to NWTSG protocols, and histologic subtypes were confirmed. Patients were assigned to the following subgroups for our data analysis: low stage (stage I and II), high stage (stage III and IV), and anaplastic. Anaplastic tumors were grouped together regardless of stage. Tumor samples were evaluated from the initial surgical resection before any adjuvant chemotherapy or radiation. Approval was obtained from the IRB at Children’s Memorial Hospital.
Tissue Processing and Immunohistochemistry Tissue samples were fixed in formalin and paraffin embedded. Sections were dried at 42°C for 24 hours and then deparaffinized, incubated at room temperature with anti-Factor VIII antibody, followed by incubation with an avidin-biotin peroxidase conjugated secondary antibody, and counterstained with hematoxylin.
Determination of Mean MVD A pathologist blinded to the stage and clinical outcome evaluated the tumor sections. The number of endothelial-lined vessels within the tumor was counted in 5 random high-powered fields (hpf) per sample. Brown staining of endothelial-lined vessels was considered positive. Values are expressed as the mean vessels per hpf ⫹/⫺ SEM.
Statistical Analysis For this study, comparisons for MVD between relapse and nonrelapse groups were examined using analysis of variance, accounting for multiple measures per patient. For subsequent analyses, the MVD values were first averaged over the 5 measurements for each case. Univariate analyses were done to relate recurrence of the WT to MVD, age, tumor weight, sex, histology, and tumor stage. Logistic regression was performed to test for significance and to obtain odds ratios and their 95% profile likelihood confidence intervals. Analyses were done on all 50 cases as well as on the 38 cases labeled as favorable histology. The MVD comparison of relapsed and nonrelapsed patients for the subsets, low-stage FH, high-stage FH, and anaplasia, was analyzed by Student’s t test utilizing the 5 MVD counts per case.
RESULTS
The mean age of the study population was 56.7 ⫹/⫺ 5.5 months with a female predominance (1.2:1). The stage and histologic distribution of the cohort was lowstage FH (n ⫽ 17), high-stage FH (n ⫽ 21), and anaplastic tumors (n ⫽ 12; 10 diffuse, 2 focal). The mean follow-up from the initial surgical resection was 3.1 ⫹/⫺ 0.71 years. Fifteen patients were identified as having tumor relapse (30%). The average time between diagnosis and relapse was 10.2 ⫹/⫺ 1.4 months, and the overall mortality rate was 8%. The sites of recurrence included the lung (n ⫽ 8), the liver/abdomen (n ⫽ 5), paraspinal (n ⫽ 1), and groin (n ⫽ 1). Histologically, the tumor sections obtained from relapsed patients appeared more hemorrhagic, and vessels were identified easily with distinct clusters of vessels infiltrating the mesenchymal component in several tumors. There was significantly higher MVD in the relapse groups versus the nonrelapse group (compare vessels in NR and R columns; Fig 1). Of particular interest is the low-stage FH group in which increased MVD correlated with recurrent disease. To establish if MVD was elevated in the relapse population (n ⫽ 15), mean MVD was compared with the nonrelapse group (n ⫽ 35) and was found to be significantly elevated (34.9 ⫹/⫺ 2.9 v 22.4 ⫹/⫺ 2; P ⬍ .05). To further stratify this group, we evaluated only the FH patients and compared the relapse (n ⫽ 13) to the nonrelapse group (n ⫽ 25). We found a significant elevation in the mean MVD (32.4 ⫹/⫺ 2.6 v 19 ⫹/⫺ 1.9; P ⬍ .05) in the relapse group. This indicates that increased angiogenesis may be a marker of more aggressive biological activity in tumors with otherwise favorable indices. To analyze the relapse and nonrelapse groups, MVD comparisons were made within the FH (low stage v high stage) and the anaplastic groups in this small pilot study, using the 5 MVD counts per patient to determine group means (Table 1). When the mean MVD per patient was used for the same comparisons, we lost significance in the high-stage FH and anaplastic groups (0.09 and 0.07, respectively), but continued to have statistical significance in the critical low stage FH subset (P ⬍ .05). To determine if MVD was a predictor of relapse in our patient population, univariate analysis was performed for each of the following parameters: age at diagnosis, tumor weight, sex, and histologic subtype (Table 2). The only factor that reached significance for the group containing all patients (n ⫽ 50) and the group evaluating only favorable histology (n ⫽ 38) was MVD (P ⬍ .05). For every increase of 10 vessels/hpf, the odds of relapse increased by a factor of 2.3 when all 50 patients were
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Fig 1. Histologic assessment of MVD in tumors stained with anti-Factor VIII antibody comparing nonrelapsed (NR) to relapsed tumors (R; original magnification ⴛ40). Dark gray staining represents endothelial lined vessels (arrows). Note the high number of vessels in all of the R tumors compared with NR.
Table 1. Comparison of MVD in Relapsed (R) and Nonrelapsed (NR) Tumors in Low-Stage, High-Stage, and Anaplastic Wilms’ Tumor Groups Tumor Stage
Relapse Status
No.
MVD
Low, FH (stage I&II) High, FH (stage III&IV) Anaplasia (all stages)
NR R NR R NR R
13 4 12 9 10 2
13.7 ⫾ 0.9 35 ⫾ 4.5 24.7 ⫾ 3.5 32.2 ⫾ 1.5 32.9 ⫾ 2.0 51 ⫾ 1.9
P Value
⬍.05 ⬍.05 ⬍.05
Abbreviations: FH, favorable histology; MVD, mean number of vessels per hpf ⫹/⫺ SEM.
analyzed and it increased by a factor of 3.93 when only the 38 patients with FH were analyzed. To make these data more clinically applicable, we tried to establish a specific value of MVD that would result in the highest sensitivity and specificity in our patient population. In assessing the total patient population, MVD of greater than 25 vessels per hpf had a sensitivity of 80% and a specificity of 66%. When evaluating only the group of patients with FH using the same MVD cut-point of 25, the sensitivity and specificity was 77% and 76%, respectively. An MVD of 25 was then applied to the low-and high-stage groups and the sensitivity, specificity, and odds ratios (OR) were calculated (Table 3). OR for this data set represents the odds of recurrent disease in patients with tumors having an
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Table 2. Results From the Univariate Analysis Variable
All Patients (n ⫽ 50) (Odds Ratio, CI)
P Value
FH Patients (n ⫽ 35) (Odds Ratio, CI)
Increase in Avg MVD by 10 vessels Increase in tumor weight by 100g Increase in age by 10 months Sex (male v female) Unfavorable histology (anaplasia v favorable)
2.31 (1.36, 4.41) 1.1 (0.90, 1.33) 1.1 (0.93, 1.27) 2.25 (0.67, 8.09) 0.39 (0.05, 1.74)
.0045* .38 .30 .20 .26
3.93 (1.8, 11.08) 1.1 (0.90, 1.37) 1.1 (0.93, 1.31) 1.48 (0.38, 6.1) N/A
P Value
.0026 .36 .28 .58
NOTE. The only variable that was predictive of relapse was MVD in both groups. Abbreviations: Avg, average; CI, confidence interval; N/A, not applicable.
MVD greater than 25 vessels per hpf relative to those below that level. Ideally, establishment of a high-risk MVD value would facilitate the development of a clinically relevant algorithm. DISCUSSION
Pathologic angiogenesis is well-documented in WT and other childhood tumors such as neuroblastoma.19 The initial goal in this pilot study was to determine if MVD could identify children with WT who have recurrent disease. Although this was a relatively small cohort of patients with a limited number of relapses, the results are encouraging with an overall 1.5-fold increased MVD in children with progressive disease. When the critical subset of patients with FH who unexpectedly relapsed was evaluated, MVD once again had predictive value. Univariate analysis confirmed that in both the overall study population and the FH group, MVD was the only variable predictive of relapse. Although this study population was limited, an MVD of 25 vessels per hpf was found to be most accurate in predicting relapse in both low- and high-stage groups supported by similar sensitivities (75% and 78%, respectively). The specificity was considerably higher in the low-stage group versus high stage (92% v 58%, respectively). It is possible that with a larger sample size, a different MVD value will need to be assigned to the high-stage group resulting in improved specificity. A new histologic marker that identifies which children are at high-risk for relapse would be exceedingly valuable, particularly for those children with otherwise favorable disease parameters. Relapse rates for children with FH Wilms’ tumor ranges from 9.6% to 22% depending on the stage at diagnosis.20 Patients with anaplasia are classified as high risk and directly placed into a more aggressive treatment arm, whereas children with Table 3. Odds Ratio Analysis Using MVD Value of 25 Vessels per hpf in Patients With Favorable Histology (FH)
All FH (n ⫽ 38) Low stage (n ⫽ 17) High stage (n ⫽ 21)
Sensitivity
Specificity
Odds Ratio
77% 75% 78%
80% 92% 58%
13.3 36 4.1
FH receive treatment based on stage alone. Whereas the overall survival rate for diffuse anaplasia remains poor, survival rates in this group have improved with specialized treatment resulting in a 4-year relapse-free survival rate now exceeding 50%.11 The ability to identify highrisk FH patients at the time of surgical resection would allow immediate intensification of treatment. At this time, there is no effective second-line therapy for treating recurrent disease, and the prognosis is dismal for this unfortunate group of children.1 Our results suggest that neovascularization in WT correlates with biological activity. The balance of angiogenic inhibitors and activators directly affects tumor vascularity, growth, and metastasis.21 Although very little is known about the effect of endogenous angiogenic inhibitors on WT growth, in vitro analysis indicates that loss of inhibitors such as thrombospondin-1 (TSP-1) may contribute to disease progression because the WT1 gene represses its expression.22 Additionally, poor prognosis has been linked to increased mutant p53 and decreased immunostaining of TSP-1 in WT.12 Support for the role of angiogenic inducers in influencing the growth of WT is considerable. Numerous inducers have been identified including vascular endothelial growth factor (VEGF), basic fibroblast growth factor, hepatocyte growth factor, and transforming growth factor beta.23,24 Further support for WT being an angiogenesis-dependent disease comes from multiple studies showing that experimental WT responds to antiangiogenic therapy with VEGF blockade,25,26 adenoviral-delivered anti–VEGF-2 receptor antagonist,27 and, most recently, topotecan.28 Evaluating the relative contributions of angiogenic mediators in vivo is a daunting task, and assessing the prognostic role of any one factor in a given tumor is even more difficult. Thus, calculating the number of blood vessels nourishing the growing tumor (MVD) more accurately reflects the net angiogenic effect within the tumor. In fact, MVD has been highly predictive of outcome in a wide variety of diseases.18 We have shown that MVD is predictive of disease outcome in WT patients. More importantly, MVD appears to be capable of identifying children with FH tumors that later present
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with progressive disease. The evaluation of MVD has several clinical advantages over some of the other proposed genetic markers. The determination requires only immunohistochemical staining, pathologic assessment, and this process results in a rapid, quantitative value that can be used for risk assignment. If the utility of MVD can be validated in a larger study, an assigned MVD value for each tumor may be integrated into
a useful algorithm for pathologists and oncologists to individualize treatment regimens. If MVD can be used in conjunction with other relevant parameters, this system potentially could affect the relapse rate and improve overall survival rate. Early and accurate classification of high-risk tumors and initiation of aggressive therapy offers the best chance of cure for children with WT.
REFERENCES 1. Pinkerton CR, Groot-Loonen JJ, Morris-Jones PH: Response rates in relapsed Wilms’ tumor. Cancer 67:567-571, 1991 2. Ghanem MA, Van der Kwast TH, Den Hollander JC, et al: Expression and prognostic value of Wilms’ tumor 1 and early growth response 1 proteins in nephroblastoma. Clin Canc Res 6:4265-4271, 2000 3. Cheah PL, Looi LM, Chan LL: Immunohistochemical expression of p53 proteins in Wilms’ tumour: A possible association with the histological prognostic parameter of anaplasia. Histopathology 28:4954, 1996 4. Beniers AJ, Efferth T, Fuzesi L, et al: p53 expression in Wilms’ tumor: A possible role as prognostic factor. In J Oncol 18:133-139, 2001 5. Sredni ST, de Camargo B, Lopes LF, et al: Immunohistochemical detection of p53 protein expression as a prognostic indicator in Wilms tumor. Med Pediatr Oncol 37:455-458, 2001 6. Mason JE, Goodfellow PJ, Grundy PE, et al: 16q loss of heterozygosity and microsatellite instability in Wilms’ tumor. J Pediatr Surg 35:891-897, 2000 7. Grundy PE, Telzerow PE, Breslow N, et al: Loss of heterozygosity for chromosomes 16q and 1p in Wilms’ tumours predicts an adverse outcome. Cancer Res 54:2331-2333, 1994 8. Grundy RG, Pritchard J, Scambler P, et al: Loss of heterozygosity on chromosome 16 in sporadic Wilms’ tumour. Br J Cancer 78:11811187, 1998 9. Klamt B, Schulze M, Thate C, et al: Allele loss in Wilms’ tumors of chromosome arms 11q, 16q and 22q correlates with clinicopathologic parameters. Genes Chromosom Cancer 22:287-294, 1998 10. Beckwith JB, Palmer NF: Histopathology and prognosis of Wilms’ tumor: Results from the first National Wilms’ Tumor Study. Cancer 41:1937-1948, 1978 11. Green DM, Beckwith JB, Breslow NE, et al: Treatment of children with stage II to IV anaplastic Wilms’ tumor: A report from the National Wilms’ Tumor Study Group. J Clin Oncol 12:2126-2131, 1994 12. Huang J, Soffer SZ, Kim ES, et al: p53 accumulation in favorable-histology Wilms’ tumor is associated with angiogenesis and clinically aggressive disease. J Pediatr Surg 37:523-527, 2002 13. Hing S, Lu Y, Summersgill B, et al: Gain of 1q is associated with adverse outcome in favorable histology Wilms’ tumors. Am J Pathol 158:393-398, 2001 14. Dome JS, Chung S, Bergemann T, et al: High telomerase reverse transcriptase (hTERT) messenger RNA level correlates with tumor
recurrence in patients with favorable histology Wilms’ tumor. Cancer Res 59:4301-4307, 1999 15. Folkman J: The role of angiogenesis in tumor growth. Semin Cancer Biol 3:65-71, 1992 16. Hanahan D, Folkman J: Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 86:353-364, 1996 17. Rowe DH, Kayton ML, O’Toole KM, et al: Pathological angiogenesis in a murine model of human Wilms’ tumor. J Pediatr Surg 34:676-679, 1999 18. Weidner N: Tumor angiogenesis: Review of current applications in tumor prognostication. Semin Diagn Pathol 10:302-313, 1993 19. Meitar D, Crawford SE, Rademaker AW, et al: Tumor angiogenesis correlates with metastatic disease, N-myc amplification, and poor outcome in human neuroblastoma. J Clin Oncol 14:405-414, 1996 20. D’Angio GJ, Breslow N, Beckwith JB, et al: Treatment of Wilms’ tumor: Results of the Third National Wilms’ Tumor Study. Cancer 64:349-360, 1989 21. Folkman J: Angiogenesis in cancer, vascular, rheumatoid, and other diseases. Nature Med 1:27-31, 1995 22. Dejong V, Degeroges A, Filleur S, et al: The Wilms’ tumor gene product represses the transcription of thrombospondin-1 in response to overexpression of c-Jun. Oncogene 18:3143-3151, 1999 23. Skoldenberg EG, Christiansson J, Sandstedt B, et al: Angiogenesis and angiogenic growth factors in Wilms’ tumor. J Urol 165:22742279, 2001 24. Kayton ML, Rowe DH, O’Toole KM, et al: Metastasis correlates with production of vascular endothelial growth factor in a murine model of human Wilms’ tumor. J Pediatr Surg 34:743-747, 1999 25. Rowe DH, Huang J, Li J, et al: Anti-VEGF antibody suppresses primary tumor growth and metastasis in an experimental model of Wilms’ tumor. J Pediatr Surg 35:30-32, 2000 26. Huang J, Moore J, Soffer S, et al: Highly specific antiangiogenic therapy is effective in suppressing growth of experimental Wilms’ tumors. J Pediatr Surg 36:357-361, 2001 27. Spurbeck WW, Ng CY, Zhou J, et al: Gene therapy-mediated expression of an angiogenesis inhibitor prevents orthotopic anaplastic Wilms’ tumor xenograft growth. Presented at the 87th Clinical Congress of the American College of Surgeons, New Orleans, LA, October, 2001 28. Soffer SZ, Kim E, Moore JT, et al: Novel use of an established agent: Topotecan is anti-angiogenic in experimental Wilms’ tumor. J Pediatr Surg 36:1781-1784, 2001
Discussion G. Gittes (Kansas City, MO): This was a very interesting study. Did you take advantage of a golden opportunity here to look at the recurrences in terms of micro-
vascular density? To understand the oncogenesis or tumor progression in this tumor, did you see a higher density in the actual relapse specimens?
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S. Crawford (response): That is a very interesting question. We only looked at 2 or 3 cases and, as you predicted, there was a higher MVD in the relapse tissue; however, perhaps when we have a larger study group we will have an opportunity to address that question more carefully. From the Floor: That was a very elegant presentation. How do you see this playing to the overall scheme of things for patients with Wilms’ tumors that do recur that are favorable stage? If we go back and look at the seminal paper by Folkman and Weidner that showed if you had breast cancer and you had increased vessels you went on to have nodal metastasis, but nothing ever evolved from that. In other words, pathologists do not look at microvasculature in breast cancer and dictate different strategies for therapy based on that. How can we make the leap in a pediatric tumor to do just that? How can we use this increased microvascular density to change therapy for Wilms’ tumor? S. Crawford (response): I think we have a unique opportunity in the pediatric group because we have a cohort of individuals that require that you provide slides and the pathologic assessment, the National Wilms’ Tumor study group (NTWTS). Something like that does not exist for the adult tumor groups. We hope that in the Wilms’ tumor study data sheet, in addition to anaplasia and determining whether it is microscopic or diffuse, there will be another category for microvascular density, perhaps with a cutoff of 25, and then on a prospective basis we will be able to evaluate whether MVD has an independent predictive value. J. Grosfeld (Indianapolis, IN): That was a very fine presentation. It is known that some of the patients with favorable histology that slip through the cracks unexpectedly and do poorly have an altered genetic makeup of their tumor; not the WT1 gene or the WT2 gene, but the first chromosome and sixteenth chromosome are abnormal. In your group of recurrences, was the genetic makeup of the tumor in those patients studied? If so, did it correlate with increased microvascular density? Did the genetic makeup have an impact on your findings?
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S. Crawford (response): Unfortunately, in our cohort of patients, because it spanned a 10-year period, it has been only recently that NTWTS has, on a regular basis, looked at p53 status and chromosomal mutations. I think as we gather data over the next 5 years, we should be able to incorporate those variables and apply some statistics to determine whether they have utility. T. Tracy (Providence, RI): I just have one quick technical question before we all run back and review all our favorable histology patients. The histology is not uniform throughout these tumors. There are different elements that show up that are more aggressive in some areas than not. How do you look at the slides and how evenly distributed is this microvascular density throughout especially the favorable histology group? S. Crawford (response): That is a very interesting question. We made sure that we avoided areas of necrosis and areas that were immediately adjacent to the kidney so that we we calculated intratumoral vessels. An interesting observation was that the mesenchymal component of the triphasic Wilms’ tumor seemed to have the highest microvascular density, although we do not have enough data to be certain about it. The blastema seems to have the lowest MVD, so we are going to try and subgroup the microvessels within the 3 phases of Wilms’ and perhaps we will be able to better answer that question. S. Soffer (Woodmere, NY): I was wondering if you had looked at p53 in your favorable-histology poor-prognosis Wilms’ tumors, because last year at APSA we presented a similar experiment in which we looked at microvascular density and we also found that there was accumulation of mutant p53 in those tumors that had a large microvascular density. There also was an upregulation of thrombospondin-1, which is an angiogenesis inhibitor. I was wondering if you had considered looking at the p53 when you do this larger study? S. Crawford (response): We would like to incorporate p53 in a future study. We read your interesting paper and it provided the background for including both p53 and thrombospondin in a larger study.
Background/Purpose: Tumor stage and histology are the most important prognostic criteria in Wilms’ tumors; however, a subset of patients remains who have favorable histology tumors and unexpectedly relapse. The authors postulated that increased microvascular density (MVD), a hallmark for angiogenesis, could identify patients at risk for relapse.
evaluating the favorable histology (FH) group alone, there was higher MVD in the relapse group (32.4 ⫹/⫺ 2.7 v 19 ⫹/⫺ 1.8; P ⬍ .05). MVD was found to be the only predictor of relapse when compared with age, sex, tumor weight, and histology.
Methods: A case-control study was used to compare relapse (n ⫽ 15) with nonrelapse tumors (n ⫽ 35). Tumor MVD was counted in 5 random high-powered fields (hpf) using antiFactor VIII antibody and expressed as mean vessel count/hpf ⫹/⫺ SEM. MVD and clinical data were evaluated using univariate analysis and student’s t test.
Conclusions: These results suggest that increased MVD can identify Wilms’ tumor patients at high risk for relapse, especially those patients with favorable histology tumors. A larger study is warranted to determine the potential utility of MVD in stratification of Wilms’ tumor patients. J Pediatr Surg 38:325-330. Copyright 2003, Elsevier Science (USA). All rights reserved.
Results: The relapse group had higher MVD than the nonrelapse group (34.9 ⫹/⫺ 2.9 v 22.4 ⫹/⫺ 2; P ⬍ .05). When
INDEX WORDS: Wilms’ tumor, microvascular density, angiogenesis, favorable histology, relapse.
W
ILMS’ TUMOR (WT) is the second most common solid abdominal tumor in childhood with overall survival rates exceeding 90%. Despite significant therapeutic advances, there still remains a subset of patients with favorable prognostic indicators who unexpectedly relapse and do not respond to aggressive second line therapy.1 This patient population continues to be the source of intensive investigation by The National Wilms’ Tumor Study Group (NWTSG), which currently is conducting its fifth study. In addition to stage, a variety of clinical and tumor-related factors have been evaluated as potential markers for poor outcome. Multivariate analysis has shown that blastemal WT suppressor gene (WT-1) expression is an independent prognostic marker for disease progression,2 whereas other studies have linked poor outcome to changes in the expression of the tumor suppressor gene p533-5 and/or loss of heterozygosity on chromosomes 16q, 1q, and 22q.6-9 The spectrum of genetic alterations in WT led to the incorporation of a prospective genetic screen in NWTSG-V. It is too early in the investigation to know if any one of these genetic indices will help identify patients at risk for recurrent disease. Despite the vast amount of genetic data accumulated from WT tissue, standard gross and histologic evaluation continues to provide important prognostic information. During NWTSG-1, anaplastic histology was found to be one of the most powerful predictors of relapse,10 and those patients with diffuse anaplasia consistently did not respond to an intensified therapeutic regimen.11 Recog-
nizing that the majority of relapsed Wilms’ tumors have favorable histology (FH), other factors clearly contribute to the aggressive phenotype. To date, one of the most difficult tasks for the oncologists has been identifying the group of patients with lower-stage FH tumors who are at risk for recurrence. When this specific subset of patients has been studied, increased p53,12 gain of 1q,13 and high telomerase reverse transcriptase (hTERT)14 were shown to positively correlate with relapse suggesting that one or more of these may prove to be useful markers. Knowing that angiogenesis is required for tumors to grow, invade, and metastasize15,16 and that pathologic angiogenesis is involved in the progression of Wilms’ tumor (WT),17 we chose to examine the predictive value of tissue microvascular density (MVD), a hallmark of angiogenesis. MVD was an important prognostic indica-
Journal of Pediatric Surgery, Vol 38, No 3 (March), 2003: pp 325-330
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From the Divisions of Pediatric Surgery and Hematology-Oncology, Children’s Memorial Hospital, Departments of Biostatistics and Pathology, Northwestern University Medical School, Chicago, IL, and the Department of Pediatrics, Cross Cancer Institute, Edmonton, Alberta, Canada. Presented at the 33rd Annual Meeting of the American Pediatric Surgical Association, Phoenix, Arizona, May 19-23, 2002. Supported by Grant CA64239 from the National Cancer Institute. Address reprint requests to Susan E. Crawford, MD, Department of Pathology, W-127, Northwestern University Medical School, 303 E Chicago Ave, Chicago, IL 60611. Copyright 2003, Elsevier Science (USA). All rights reserved. 0022-3468/03/3803-0010$35.00/0 doi:10.1053/jpsu.2003.50102
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tor in a wide variety of carcinomas including breast, prostate, and non–small cell lung.18 This pilot study sought to determine if higher MVD correlated with more aggressive biological behavior in Wilms’ tumor patients. Furthermore, we evaluated the subgroup of patients with FH to determine if MVD may be able to identify those children without adverse prognostic features who relapsed. If MVD had predictive value, this would allow earlier intensive treatment and heightened surveillance in patients with highly vascular tumors. MATERIALS AND METHODS
Sample Selection and Tissue Acquisition A case-control study was used to compare biological differences in tumors from patients with and without relapsed Wilms’ tumors. A review of all WT patients treated at a single institution from 1981 through 2001 (n ⫽ 88) was undertaken to identify relapse patients (n ⫽ 12). Three additional cases of relapsed patients were obtained from the NWTSG tissue bank. Nonrelapsed controls were obtained from both the NWTSG and the Children’s Memorial Hospital with a minimum disease-free follow-up of 2 years (n ⫽ 35). Patients were staged clinically and treated according to NWTSG protocols, and histologic subtypes were confirmed. Patients were assigned to the following subgroups for our data analysis: low stage (stage I and II), high stage (stage III and IV), and anaplastic. Anaplastic tumors were grouped together regardless of stage. Tumor samples were evaluated from the initial surgical resection before any adjuvant chemotherapy or radiation. Approval was obtained from the IRB at Children’s Memorial Hospital.
Tissue Processing and Immunohistochemistry Tissue samples were fixed in formalin and paraffin embedded. Sections were dried at 42°C for 24 hours and then deparaffinized, incubated at room temperature with anti-Factor VIII antibody, followed by incubation with an avidin-biotin peroxidase conjugated secondary antibody, and counterstained with hematoxylin.
Determination of Mean MVD A pathologist blinded to the stage and clinical outcome evaluated the tumor sections. The number of endothelial-lined vessels within the tumor was counted in 5 random high-powered fields (hpf) per sample. Brown staining of endothelial-lined vessels was considered positive. Values are expressed as the mean vessels per hpf ⫹/⫺ SEM.
Statistical Analysis For this study, comparisons for MVD between relapse and nonrelapse groups were examined using analysis of variance, accounting for multiple measures per patient. For subsequent analyses, the MVD values were first averaged over the 5 measurements for each case. Univariate analyses were done to relate recurrence of the WT to MVD, age, tumor weight, sex, histology, and tumor stage. Logistic regression was performed to test for significance and to obtain odds ratios and their 95% profile likelihood confidence intervals. Analyses were done on all 50 cases as well as on the 38 cases labeled as favorable histology. The MVD comparison of relapsed and nonrelapsed patients for the subsets, low-stage FH, high-stage FH, and anaplasia, was analyzed by Student’s t test utilizing the 5 MVD counts per case.
RESULTS
The mean age of the study population was 56.7 ⫹/⫺ 5.5 months with a female predominance (1.2:1). The stage and histologic distribution of the cohort was lowstage FH (n ⫽ 17), high-stage FH (n ⫽ 21), and anaplastic tumors (n ⫽ 12; 10 diffuse, 2 focal). The mean follow-up from the initial surgical resection was 3.1 ⫹/⫺ 0.71 years. Fifteen patients were identified as having tumor relapse (30%). The average time between diagnosis and relapse was 10.2 ⫹/⫺ 1.4 months, and the overall mortality rate was 8%. The sites of recurrence included the lung (n ⫽ 8), the liver/abdomen (n ⫽ 5), paraspinal (n ⫽ 1), and groin (n ⫽ 1). Histologically, the tumor sections obtained from relapsed patients appeared more hemorrhagic, and vessels were identified easily with distinct clusters of vessels infiltrating the mesenchymal component in several tumors. There was significantly higher MVD in the relapse groups versus the nonrelapse group (compare vessels in NR and R columns; Fig 1). Of particular interest is the low-stage FH group in which increased MVD correlated with recurrent disease. To establish if MVD was elevated in the relapse population (n ⫽ 15), mean MVD was compared with the nonrelapse group (n ⫽ 35) and was found to be significantly elevated (34.9 ⫹/⫺ 2.9 v 22.4 ⫹/⫺ 2; P ⬍ .05). To further stratify this group, we evaluated only the FH patients and compared the relapse (n ⫽ 13) to the nonrelapse group (n ⫽ 25). We found a significant elevation in the mean MVD (32.4 ⫹/⫺ 2.6 v 19 ⫹/⫺ 1.9; P ⬍ .05) in the relapse group. This indicates that increased angiogenesis may be a marker of more aggressive biological activity in tumors with otherwise favorable indices. To analyze the relapse and nonrelapse groups, MVD comparisons were made within the FH (low stage v high stage) and the anaplastic groups in this small pilot study, using the 5 MVD counts per patient to determine group means (Table 1). When the mean MVD per patient was used for the same comparisons, we lost significance in the high-stage FH and anaplastic groups (0.09 and 0.07, respectively), but continued to have statistical significance in the critical low stage FH subset (P ⬍ .05). To determine if MVD was a predictor of relapse in our patient population, univariate analysis was performed for each of the following parameters: age at diagnosis, tumor weight, sex, and histologic subtype (Table 2). The only factor that reached significance for the group containing all patients (n ⫽ 50) and the group evaluating only favorable histology (n ⫽ 38) was MVD (P ⬍ .05). For every increase of 10 vessels/hpf, the odds of relapse increased by a factor of 2.3 when all 50 patients were
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Fig 1. Histologic assessment of MVD in tumors stained with anti-Factor VIII antibody comparing nonrelapsed (NR) to relapsed tumors (R; original magnification ⴛ40). Dark gray staining represents endothelial lined vessels (arrows). Note the high number of vessels in all of the R tumors compared with NR.
Table 1. Comparison of MVD in Relapsed (R) and Nonrelapsed (NR) Tumors in Low-Stage, High-Stage, and Anaplastic Wilms’ Tumor Groups Tumor Stage
Relapse Status
No.
MVD
Low, FH (stage I&II) High, FH (stage III&IV) Anaplasia (all stages)
NR R NR R NR R
13 4 12 9 10 2
13.7 ⫾ 0.9 35 ⫾ 4.5 24.7 ⫾ 3.5 32.2 ⫾ 1.5 32.9 ⫾ 2.0 51 ⫾ 1.9
P Value
⬍.05 ⬍.05 ⬍.05
Abbreviations: FH, favorable histology; MVD, mean number of vessels per hpf ⫹/⫺ SEM.
analyzed and it increased by a factor of 3.93 when only the 38 patients with FH were analyzed. To make these data more clinically applicable, we tried to establish a specific value of MVD that would result in the highest sensitivity and specificity in our patient population. In assessing the total patient population, MVD of greater than 25 vessels per hpf had a sensitivity of 80% and a specificity of 66%. When evaluating only the group of patients with FH using the same MVD cut-point of 25, the sensitivity and specificity was 77% and 76%, respectively. An MVD of 25 was then applied to the low-and high-stage groups and the sensitivity, specificity, and odds ratios (OR) were calculated (Table 3). OR for this data set represents the odds of recurrent disease in patients with tumors having an
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Table 2. Results From the Univariate Analysis Variable
All Patients (n ⫽ 50) (Odds Ratio, CI)
P Value
FH Patients (n ⫽ 35) (Odds Ratio, CI)
Increase in Avg MVD by 10 vessels Increase in tumor weight by 100g Increase in age by 10 months Sex (male v female) Unfavorable histology (anaplasia v favorable)
2.31 (1.36, 4.41) 1.1 (0.90, 1.33) 1.1 (0.93, 1.27) 2.25 (0.67, 8.09) 0.39 (0.05, 1.74)
.0045* .38 .30 .20 .26
3.93 (1.8, 11.08) 1.1 (0.90, 1.37) 1.1 (0.93, 1.31) 1.48 (0.38, 6.1) N/A
P Value
.0026 .36 .28 .58
NOTE. The only variable that was predictive of relapse was MVD in both groups. Abbreviations: Avg, average; CI, confidence interval; N/A, not applicable.
MVD greater than 25 vessels per hpf relative to those below that level. Ideally, establishment of a high-risk MVD value would facilitate the development of a clinically relevant algorithm. DISCUSSION
Pathologic angiogenesis is well-documented in WT and other childhood tumors such as neuroblastoma.19 The initial goal in this pilot study was to determine if MVD could identify children with WT who have recurrent disease. Although this was a relatively small cohort of patients with a limited number of relapses, the results are encouraging with an overall 1.5-fold increased MVD in children with progressive disease. When the critical subset of patients with FH who unexpectedly relapsed was evaluated, MVD once again had predictive value. Univariate analysis confirmed that in both the overall study population and the FH group, MVD was the only variable predictive of relapse. Although this study population was limited, an MVD of 25 vessels per hpf was found to be most accurate in predicting relapse in both low- and high-stage groups supported by similar sensitivities (75% and 78%, respectively). The specificity was considerably higher in the low-stage group versus high stage (92% v 58%, respectively). It is possible that with a larger sample size, a different MVD value will need to be assigned to the high-stage group resulting in improved specificity. A new histologic marker that identifies which children are at high-risk for relapse would be exceedingly valuable, particularly for those children with otherwise favorable disease parameters. Relapse rates for children with FH Wilms’ tumor ranges from 9.6% to 22% depending on the stage at diagnosis.20 Patients with anaplasia are classified as high risk and directly placed into a more aggressive treatment arm, whereas children with Table 3. Odds Ratio Analysis Using MVD Value of 25 Vessels per hpf in Patients With Favorable Histology (FH)
All FH (n ⫽ 38) Low stage (n ⫽ 17) High stage (n ⫽ 21)
Sensitivity
Specificity
Odds Ratio
77% 75% 78%
80% 92% 58%
13.3 36 4.1
FH receive treatment based on stage alone. Whereas the overall survival rate for diffuse anaplasia remains poor, survival rates in this group have improved with specialized treatment resulting in a 4-year relapse-free survival rate now exceeding 50%.11 The ability to identify highrisk FH patients at the time of surgical resection would allow immediate intensification of treatment. At this time, there is no effective second-line therapy for treating recurrent disease, and the prognosis is dismal for this unfortunate group of children.1 Our results suggest that neovascularization in WT correlates with biological activity. The balance of angiogenic inhibitors and activators directly affects tumor vascularity, growth, and metastasis.21 Although very little is known about the effect of endogenous angiogenic inhibitors on WT growth, in vitro analysis indicates that loss of inhibitors such as thrombospondin-1 (TSP-1) may contribute to disease progression because the WT1 gene represses its expression.22 Additionally, poor prognosis has been linked to increased mutant p53 and decreased immunostaining of TSP-1 in WT.12 Support for the role of angiogenic inducers in influencing the growth of WT is considerable. Numerous inducers have been identified including vascular endothelial growth factor (VEGF), basic fibroblast growth factor, hepatocyte growth factor, and transforming growth factor beta.23,24 Further support for WT being an angiogenesis-dependent disease comes from multiple studies showing that experimental WT responds to antiangiogenic therapy with VEGF blockade,25,26 adenoviral-delivered anti–VEGF-2 receptor antagonist,27 and, most recently, topotecan.28 Evaluating the relative contributions of angiogenic mediators in vivo is a daunting task, and assessing the prognostic role of any one factor in a given tumor is even more difficult. Thus, calculating the number of blood vessels nourishing the growing tumor (MVD) more accurately reflects the net angiogenic effect within the tumor. In fact, MVD has been highly predictive of outcome in a wide variety of diseases.18 We have shown that MVD is predictive of disease outcome in WT patients. More importantly, MVD appears to be capable of identifying children with FH tumors that later present
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with progressive disease. The evaluation of MVD has several clinical advantages over some of the other proposed genetic markers. The determination requires only immunohistochemical staining, pathologic assessment, and this process results in a rapid, quantitative value that can be used for risk assignment. If the utility of MVD can be validated in a larger study, an assigned MVD value for each tumor may be integrated into
a useful algorithm for pathologists and oncologists to individualize treatment regimens. If MVD can be used in conjunction with other relevant parameters, this system potentially could affect the relapse rate and improve overall survival rate. Early and accurate classification of high-risk tumors and initiation of aggressive therapy offers the best chance of cure for children with WT.
REFERENCES 1. Pinkerton CR, Groot-Loonen JJ, Morris-Jones PH: Response rates in relapsed Wilms’ tumor. Cancer 67:567-571, 1991 2. Ghanem MA, Van der Kwast TH, Den Hollander JC, et al: Expression and prognostic value of Wilms’ tumor 1 and early growth response 1 proteins in nephroblastoma. Clin Canc Res 6:4265-4271, 2000 3. Cheah PL, Looi LM, Chan LL: Immunohistochemical expression of p53 proteins in Wilms’ tumour: A possible association with the histological prognostic parameter of anaplasia. Histopathology 28:4954, 1996 4. Beniers AJ, Efferth T, Fuzesi L, et al: p53 expression in Wilms’ tumor: A possible role as prognostic factor. In J Oncol 18:133-139, 2001 5. Sredni ST, de Camargo B, Lopes LF, et al: Immunohistochemical detection of p53 protein expression as a prognostic indicator in Wilms tumor. Med Pediatr Oncol 37:455-458, 2001 6. Mason JE, Goodfellow PJ, Grundy PE, et al: 16q loss of heterozygosity and microsatellite instability in Wilms’ tumor. J Pediatr Surg 35:891-897, 2000 7. Grundy PE, Telzerow PE, Breslow N, et al: Loss of heterozygosity for chromosomes 16q and 1p in Wilms’ tumours predicts an adverse outcome. Cancer Res 54:2331-2333, 1994 8. Grundy RG, Pritchard J, Scambler P, et al: Loss of heterozygosity on chromosome 16 in sporadic Wilms’ tumour. Br J Cancer 78:11811187, 1998 9. Klamt B, Schulze M, Thate C, et al: Allele loss in Wilms’ tumors of chromosome arms 11q, 16q and 22q correlates with clinicopathologic parameters. Genes Chromosom Cancer 22:287-294, 1998 10. Beckwith JB, Palmer NF: Histopathology and prognosis of Wilms’ tumor: Results from the first National Wilms’ Tumor Study. Cancer 41:1937-1948, 1978 11. Green DM, Beckwith JB, Breslow NE, et al: Treatment of children with stage II to IV anaplastic Wilms’ tumor: A report from the National Wilms’ Tumor Study Group. J Clin Oncol 12:2126-2131, 1994 12. Huang J, Soffer SZ, Kim ES, et al: p53 accumulation in favorable-histology Wilms’ tumor is associated with angiogenesis and clinically aggressive disease. J Pediatr Surg 37:523-527, 2002 13. Hing S, Lu Y, Summersgill B, et al: Gain of 1q is associated with adverse outcome in favorable histology Wilms’ tumors. Am J Pathol 158:393-398, 2001 14. Dome JS, Chung S, Bergemann T, et al: High telomerase reverse transcriptase (hTERT) messenger RNA level correlates with tumor
recurrence in patients with favorable histology Wilms’ tumor. Cancer Res 59:4301-4307, 1999 15. Folkman J: The role of angiogenesis in tumor growth. Semin Cancer Biol 3:65-71, 1992 16. Hanahan D, Folkman J: Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 86:353-364, 1996 17. Rowe DH, Kayton ML, O’Toole KM, et al: Pathological angiogenesis in a murine model of human Wilms’ tumor. J Pediatr Surg 34:676-679, 1999 18. Weidner N: Tumor angiogenesis: Review of current applications in tumor prognostication. Semin Diagn Pathol 10:302-313, 1993 19. Meitar D, Crawford SE, Rademaker AW, et al: Tumor angiogenesis correlates with metastatic disease, N-myc amplification, and poor outcome in human neuroblastoma. J Clin Oncol 14:405-414, 1996 20. D’Angio GJ, Breslow N, Beckwith JB, et al: Treatment of Wilms’ tumor: Results of the Third National Wilms’ Tumor Study. Cancer 64:349-360, 1989 21. Folkman J: Angiogenesis in cancer, vascular, rheumatoid, and other diseases. Nature Med 1:27-31, 1995 22. Dejong V, Degeroges A, Filleur S, et al: The Wilms’ tumor gene product represses the transcription of thrombospondin-1 in response to overexpression of c-Jun. Oncogene 18:3143-3151, 1999 23. Skoldenberg EG, Christiansson J, Sandstedt B, et al: Angiogenesis and angiogenic growth factors in Wilms’ tumor. J Urol 165:22742279, 2001 24. Kayton ML, Rowe DH, O’Toole KM, et al: Metastasis correlates with production of vascular endothelial growth factor in a murine model of human Wilms’ tumor. J Pediatr Surg 34:743-747, 1999 25. Rowe DH, Huang J, Li J, et al: Anti-VEGF antibody suppresses primary tumor growth and metastasis in an experimental model of Wilms’ tumor. J Pediatr Surg 35:30-32, 2000 26. Huang J, Moore J, Soffer S, et al: Highly specific antiangiogenic therapy is effective in suppressing growth of experimental Wilms’ tumors. J Pediatr Surg 36:357-361, 2001 27. Spurbeck WW, Ng CY, Zhou J, et al: Gene therapy-mediated expression of an angiogenesis inhibitor prevents orthotopic anaplastic Wilms’ tumor xenograft growth. Presented at the 87th Clinical Congress of the American College of Surgeons, New Orleans, LA, October, 2001 28. Soffer SZ, Kim E, Moore JT, et al: Novel use of an established agent: Topotecan is anti-angiogenic in experimental Wilms’ tumor. J Pediatr Surg 36:1781-1784, 2001
Discussion G. Gittes (Kansas City, MO): This was a very interesting study. Did you take advantage of a golden opportunity here to look at the recurrences in terms of micro-
vascular density? To understand the oncogenesis or tumor progression in this tumor, did you see a higher density in the actual relapse specimens?
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S. Crawford (response): That is a very interesting question. We only looked at 2 or 3 cases and, as you predicted, there was a higher MVD in the relapse tissue; however, perhaps when we have a larger study group we will have an opportunity to address that question more carefully. From the Floor: That was a very elegant presentation. How do you see this playing to the overall scheme of things for patients with Wilms’ tumors that do recur that are favorable stage? If we go back and look at the seminal paper by Folkman and Weidner that showed if you had breast cancer and you had increased vessels you went on to have nodal metastasis, but nothing ever evolved from that. In other words, pathologists do not look at microvasculature in breast cancer and dictate different strategies for therapy based on that. How can we make the leap in a pediatric tumor to do just that? How can we use this increased microvascular density to change therapy for Wilms’ tumor? S. Crawford (response): I think we have a unique opportunity in the pediatric group because we have a cohort of individuals that require that you provide slides and the pathologic assessment, the National Wilms’ Tumor study group (NTWTS). Something like that does not exist for the adult tumor groups. We hope that in the Wilms’ tumor study data sheet, in addition to anaplasia and determining whether it is microscopic or diffuse, there will be another category for microvascular density, perhaps with a cutoff of 25, and then on a prospective basis we will be able to evaluate whether MVD has an independent predictive value. J. Grosfeld (Indianapolis, IN): That was a very fine presentation. It is known that some of the patients with favorable histology that slip through the cracks unexpectedly and do poorly have an altered genetic makeup of their tumor; not the WT1 gene or the WT2 gene, but the first chromosome and sixteenth chromosome are abnormal. In your group of recurrences, was the genetic makeup of the tumor in those patients studied? If so, did it correlate with increased microvascular density? Did the genetic makeup have an impact on your findings?
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S. Crawford (response): Unfortunately, in our cohort of patients, because it spanned a 10-year period, it has been only recently that NTWTS has, on a regular basis, looked at p53 status and chromosomal mutations. I think as we gather data over the next 5 years, we should be able to incorporate those variables and apply some statistics to determine whether they have utility. T. Tracy (Providence, RI): I just have one quick technical question before we all run back and review all our favorable histology patients. The histology is not uniform throughout these tumors. There are different elements that show up that are more aggressive in some areas than not. How do you look at the slides and how evenly distributed is this microvascular density throughout especially the favorable histology group? S. Crawford (response): That is a very interesting question. We made sure that we avoided areas of necrosis and areas that were immediately adjacent to the kidney so that we we calculated intratumoral vessels. An interesting observation was that the mesenchymal component of the triphasic Wilms’ tumor seemed to have the highest microvascular density, although we do not have enough data to be certain about it. The blastema seems to have the lowest MVD, so we are going to try and subgroup the microvessels within the 3 phases of Wilms’ and perhaps we will be able to better answer that question. S. Soffer (Woodmere, NY): I was wondering if you had looked at p53 in your favorable-histology poor-prognosis Wilms’ tumors, because last year at APSA we presented a similar experiment in which we looked at microvascular density and we also found that there was accumulation of mutant p53 in those tumors that had a large microvascular density. There also was an upregulation of thrombospondin-1, which is an angiogenesis inhibitor. I was wondering if you had considered looking at the p53 when you do this larger study? S. Crawford (response): We would like to incorporate p53 in a future study. We read your interesting paper and it provided the background for including both p53 and thrombospondin in a larger study.