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© 2000 American Society for Clinical Oncology Angiogenesis in Neuroblastoma: Relationship to Survival and Other Prognostic Factors in a Cohort of Neuroblastoma PatientsFrom the Pediatric Oncology Unit, Hospital Infantil La Fe; Department of Pathology, Medical School; and the Biostatistics Department, University of Valencia, Valencia, Spain. Address reprint requests to Adela Cañete, MD, Pediatric Oncology Unit, Hospital Infantil La Fe, Avda Campanar 21, 46009 Valencia, Spain; email anieto{at}ene.es
PURPOSE: To study angiogenesis in neuroblastoma, using morphometric and computerized image analysis, and correlate the results with survival and other prognostic factors. PATIENTS AND METHODS: Sixty-nine patients from the Spanish Cooperative Study for Neuroblastoma were studied. Tumoral angiogenesis was studied using an avidin-biotin immunoperoxidase technique with an anti-CD34 antibody. Vascular parameters (VPs) were analyzed by a computerized system. Statistical analysis was also performed. RESULTS: Sixty-six samples had adequate tumoral tissue, and their tumoral vessels were counted. Endothelial cells were more prominent in pure neuroblastomas than in maturing and more mature tumors. VPs showed no statistical difference between the groups of patients as defined by the levels of the other prognostic factors in neuroblastoma: age, stage, histopathology, TRK-A, P-glycoprotein expression, or MYCN copy number. In patients who relapsed, tumors did not show statistically significant difference in VPs when compared with tumors from patients who did not relapse. There was also no difference in VPs in tumors from living patients when compared with tumors from deceased patients. Overall survival was 75%, and event-free survival was 55% at 50 months. CONCLUSION: VPs could be adequately determined by a computerized system in neuroblastoma; however, VPs were not predictive of survival for our patients. In our patients, neither disseminated nor local relapses were influenced by the angiogenic characteristics of the tumors.
NEUROBLASTOMA (NB) is the second most common solid tumor in childhood. It is an embryonal neoplasm derived from the neural crest, and its biologic behavior is quite intriguing: some of these tumors disappear with very little or no treatment, whereas other patients die despite undergoing treatment with aggressive therapies.1 Pediatric oncologists try to identify those patients who need such aggressive treatments and those who do not. Age,2 stage,3,4 and MYCN amplification5-8 are prognostic factors that clearly influence survival; loss of heterozygosity in the short arm of chromosome 1,9-12 gains in chromosome 17,11,13,14 and other genetic factors15,16 are emerging as important prognostic factors in survival and relapse-free status. Secondary prognostic factors such as TRK-A,17 CD44,18 P-glycoprotein,19 and bcl-2 protein expression20 have also been reported. Tumor growth depends not only on the mitotic index of the proliferating cells, but also on complex phenomena such as angiogenesis. Folkman et al21 in 1971 demonstrated the mechanisms and relevance of angiogenesis in malignant tumors, and, since then, it has been related to prognosis in many adult cancers.22-36 Studies in pediatric populations are scarce. Recently, Meitar et al37 analyzed the phenomenon of angiogenesis in NB patients, and a high vascular index ( 4) was correlated with disseminated disease, MYCN amplification, unfavorable histology, and low survival. Therefore, we performed a study to determine whether angiogenesis predicts adverse outcome in patients with NB. There are several methods to measure the angiogenesis in neoplasms, but immunohistochemistry seems to be the mainstay of all. Monoclonal antibodies to endothelial cell markers are readily available,38-41 although some do not work properly on paraffin sections. Likewise, vessel quantification can be performed either manually or by computerized systems.42,43 Discrepancies in various studies have demonstrated the need for a consensus and a standardization in the methodology for vessel staining and counting. These aspects have recently been published.44
Patients from the second Spanish Neuroblastoma Cooperative Study (N-II-92) were studied. Twenty-six Spanish hospitals participated in this protocol (see Appendix). Eligibility criteria for entry onto N-II-92 included age (children between 1 day and 15 years of age were eligible), histologically proven diagnosis of NB in either primary or metastatic tumor, no previous chemotherapy, and normal renal function (glomerular filtration rate 80 mL/min/1.73 m2). All eligible patients were registered. Clinical data regarding age, stage (according to the International Neuroblastoma Staging System), location of the tumor, and biologic factors (catecholamine metabolites, enolase, ferritin, lactate dehydrogenase) were sent to a centralized office located at the Pediatric Oncology Unit in La Fe Children’s Hospital, Valencia, Spain. Response to therapy, survival, adverse events, and follow-up were also registered there. At diagnosis, before chemotherapy treatment was initiated, frozen and fresh tumor specimens, paraffin blocks, and peripheral-blood samples were sent to the reference laboratory for biologic studies at the Department of Pathology (Medical School, University of Valencia, Valencia, Spain). These studies and the methods used are described briefly: histologic classification was performed according to Shimada et al,45 MYCN amplification was determined using Southern blot analysis 46 and polymerase chain reaction,47 ploidy was examined by static cytometry after Feulgen staining,48 and P-glycoprotein expression19 and TRK49 expression were studied using standard immunohistochemical techniques. Patients were treated according to stage and MYCN amplification (defined as more than 10 copies of the oncogene): stage 1 patients were treated only with surgery. Between 1992 and 1994, 13 stage 2 patients received mild chemotherapy after surgery; and from 1995, they were treated only with surgery, according to a European cooperative study. Stage 3 and 4 patients received induction chemotherapy comprising high-dose cyclophosphamide, carboplatin, cisplatin, vincristine, teniposide, and etoposide. After surgery, stage 4 patients were treated with megatherapy and stem-cell transplantation, providing that they were in complete or partial remission. Stage 3 patients were treated with maintenance chemotherapy (cyclophosphamide, epiadriamycin, vincristine, dacarbazine, carboplatin, and etoposide) for 9 months. Radiotherapy was administered in cases of incomplete resection of primary tumor. Stage 4 infants were treated with the same induction chemotherapy that was administered to children older than 1 year of age (although the regimen was shorter and lower doses were used), followed by maintenance chemotherapy after surgery. Patients with MYCN amplification were treated in the same manner as stage 4 patients. Angiogenesis studies were designed in 1996, and eligibility was set up according to chronologic criteria: 69 consecutive patients who had enough and adequate tumor material to perform all of the biologic and histologic studies were initially chosen. The method for staining tumor vessels (including the choice of antibody and the image analysis) was performed according to the techniques used in the Department of Pathology (Medical School, University of Valencia) to study angiogenesis in breast cancer and other adult tumors.50 Tumoral angiogenesis was studied on 5-µm paraffin sections using an avidin-biotin immunoperoxidase technique, as described by Hsu et al,51 with few modifications: after fixation and endogenous peroxidase blockage, sections were immersed in citrate tampon (0.1 mol/L, pH6), which was pretreated with three microwave shocks and enzymatic digestion with Pronase E (1:1000) for 5 minutes to facilitate antigen retrieval. The first antibody was a mouse monoclonal, anti-CD34 (Biogenex, San Ramon, CA), used at 1:20 and incubated overnight in a humid chamber. After three washes with 1 x phosphate-buffered saline, an antimouse IgG with peroxidase and biotin (Vector Laboratories, Inc, Burlingame, CA) was applied at 1:200 dilution (in 10% horse serum) for 30 minutes. Simultaneously, avidin-biotin complexes (Vectastain Kit, Vector Laboratories) were prepared according to the manufacturer’s instructions. After a 30-minute incubation with the secondary antibody, three 1 x phosphate-buffered saline washes were carried out and the avidin-biotin complexes applied. After three new washes, diaminobenzidine (0.5 mg/mL) and imidazole52 (0.06 mg/mL) in Trisma pH 7 plus 35 µL of hydrogen peroxide (in 100 mL of chromogen) were applied, at 37°C in darkness, for 2 minutes. Counterstaining was performed using methylene green. Nontumoral vessels were used as internal positive controls as described by Siitonen et al.53 Angiogenesis parameters were assessed in the richest vascular area of each sample (hot spot), chosen at low magnification (x40) as described by Weidner et al.23 Then, image analysis was performed by a computerized system, CUE-2 (Olympus, Tokyo, Japan), which consisted of special software, image processor, CCD camera, and video monitor connected to an Olympus microscope (BH-2). This image analysis was performed at higher magnification (x200) on 10 images for each selected area in one sample, with a final analyzed area of 0.59 mm2 in each tumor. To increase the contrast between brown from endothelial cells (obtained by diamino benzidine staining) and green from the nuclei (obtained with the methylene green), a 436-nm filter (Olympus) was used in the microscope. The image analysis was performed with the computer54,55: images were captured using the microscope coupled to a black and white video camera and digitized using an internal frame-grabbing board and Apple computer (Apple Computer, Inc, Cupertino, CA). This last procedure consisted of converting the captured image in points or pixels according to the gray tone. Once the computer selected these areas of similar color (within a set of tolerance and after eliminating the background), these regions were filled in black and the image was converted to a gray scale. According to Bosari et al,56 vessels larger than 50 µm and those with muscular coat were excluded. The computerized system measured the following in each of the 10 areas: microvessel number (the number of vessels per mm2 derived from image analysis), luminal area, vessel perimeter, vessel volume, vessel density, and vessel diameter. Finally, it integrated the results from the 10 areas into one single result of each parameter for a single sample. These final data on vascular area, vascular density, number of vessels, vascular perimeter, diameter of the vessels, and vascular volume for each tumor were saved in files and histograms.
Statistical analysis was performed using SPSS 6.01 software (SPSS, Inc, Chicago, IL). The following tests were applied:
A total of 206 patients were registered in the N-II-92 protocol between June 1992 and June 1997. Sixty-nine of these patients were initially studied for angiogenesis, although adequate material was obtained in only 66 patients, who represent the cases analyzed in this article. All patients were followed-up. Clinical, biologic, and genetic characteristics of both groups (those registered in the N-II-92 protocol and those studied for angiogenesis) are listed in Table 1.
Anti-CD34 Staining Sixty-six samples, all obtained at diagnosis, had adequate tumoral tissue, and newly formed tumoral vessels were identified and counted according to the method described under Patients and Methods. Morphologically, newly formed vessels were abundant, with smaller vascular diameter and without muscular wall, when compared with normal vessels. Endothelial cells were more prominent and stained in pure NBs than in maturing and more mature tumors. Neither neuroblastic cells nor Schwann cells showed any positive staining with the anti-CD34 antibody. These characteristics are shown in Figs 1 and 2. Morphometric data were also calculated, and the range, mean, and median of the vascular parameters are listed in Table 2.
Relationship With Other Prognostic Factors The existence of a possible relationship of these vascular parameters with other well-known prognostic factors in NB was studied by nonparametric tests (Table 3): there was no statistical difference in any of the vascular parameters studied according to age (older than and younger than 1 year of age), different disease stages, favorable versus unfavorable histopathology, TRK-A, P-glycoprotein expression, and MYCN-amplified cases, although there were only three patients who were considered to have amplification of the MYCN oncogene (with more than 10 copies of the oncogene). The mean values of the vascular parameters in these patients are listed in Table 4, as compared with the mean values of MYCN-normal patients.
Vascular Parameters and Relapses There were 18 relapses (11 disseminated and seven exclusively local). When we studied the vascular parameters in the different types of relapses, it was observed that there were no differences in any of the vascular parameters between tumors obtained from patients who relapsed locally and tumors obtained from those patients who did not relapse. In disseminated relapses, newly formed vessels were smaller in size, vascular perimeter and area were also smaller, and there were more abundant vessels, but the differences were not statistically significant (Table 5).
Survival in the Angiogenesis Group Fourteen patients died, 12 from progression of their disease. Six patients were lost to follow-up at 2, 3, 4, 10, and 13 months from diagnosis. Overall survival (OS) for our patients was 75%, and event-free survival (EFS) was 55% at 50 months (95% confidence interval, 38.19% to 72.57%), with a median follow-up of 54 months (Fig 3). Stepwise selection of variables in Cox regression showed that enolase and Shimada classification are the most important prognostic factors in predicting survival in this cohort of patients. Table 6 lists the first step of that sequential procedure. When vascular parameters were studied, they did not show any difference between living and dead patients in any of the vascular parameters, as listed in Table 3.
Angiogenesis has been widely studied in different adult neoplasms57: in breast cancer, 69% to 80% of the studies with a multivariate analysis found that intratumoral microvessel density (IMD) is of prognostic value for survival (OS and relapse-free survival). In non–small-cell lung cancer, it has been shown that there is a statistically significant association between vascularity and metastasis. In gastrointestinal tumors, expression of vascular endothelial growth factor and IMD are both associated and predictive of recurrence. In female and male urogenital cancers, angiogenesis also seems to be important. Surprisingly, in pediatric malignancies—which are highly vascularized and quite aggressive—published studies involving angiogenesis are scarce. There are few retrospective studies about the prognostic significance of angiogenesis,37 there are no prospective studies, and there are few that deal with future antiangiogenic therapy.58,59 Although angiogenesis might have an impact on many pediatric cancers, we decided to study NB for three reasons: (1) it exhibits intriguing biologic behavior, (2) a cooperative study with good accrual and a centralized study of biologic factors was underway, and (3) a previous report37 on the relevance of angiogenesis in NB had already been published. However, this is the first time that different vascular parameters have been defined in a group of NB patients from a national cooperative study group by means of a computerized image analysis. We chose this methodology because we found it to be more objective and reproducible. We decided not to define a vascular index in our samples, but to study the newly formed tumoral vessels in a descriptive way and to correlate our results with prognosis and survival. Two technical problems were faced: (1) election of endothelial cell marker, and (2) election of the method of counting vessels. For the first point, we chose an anti-CD34 antibody as endothelial cell marker on the basis of studies in breast cancer in our laboratory.50 It is well known that there is no "ideal" endothelial cell marker for angiogenesis; all of the existing antibodies have disadvantages, above all, in working with paraffin sections. Previous studies in our laboratory compared antifactor VIII, anti-CD31, and anti-CD34 in breast cancer, and the results showed that anti-CD34 stained newly formed tumoral vessels (without muscular coat) properly, with a very weak background staining, even for vessels with muscular coat.50 To facilitate antibody penetration in the samples, we modified the standard immunohistochemical technique (as described in Patients and Methods) to assure an optimal reading with the image analyzer. With these modifications, all of our samples could be analyzed adequately. For the second point, we chose to perform a computer image analysis. Manual methods are diverse and broadly used, as they do not need a complex procedure to be performed (they can be carried out on hematoxylin-eosin sections, with optic microscope, and without computer assistance). There are several methods: manual counting of microvessels in more vascularized areas in a tumor by the investigator, gradation of IMD (as described by Weidner et al23 and Guidi et al60 ), and microvessel counting according to Chalkley.42 All of these methods determine only microvessel numbers or artificially created vascular index (relating number of microvessels and tumoral area), with different cutoff points. Furthermore, they can be subjected to interobserver variability. Computer systems have the advantage that they can analyze not only the number of microvessels but also other vascular parameters (such as area, volume, or density) without direct intervention of the investigator. With the great amount of basic and clinical research on angiogenesis nowadays, quantification of this phenomenon in solid tumors remains controversial, despite an International Consensus in 1996. We agree with authors such as Vermeulen et al44 and Ellis,61 who pointed out the need to define more refined, objective, and accurate methods to quantify angiogenesis in order to incorporate this parameter in prospective studies. Although this is a retrospective study, all patients were included in the current study of the Spanish Society of Pediatric Oncology and were diagnosed and treated according thereto. Other authors have also retrospectively studied angiogenesis in NB patients treated at a single institution over a long period of time, with differences in outcome that could be influenced not only by biologic factors but also by different therapeutic strategies that have been used over the years. There are other differences between our study and that of Meitar et al37 that make them noncomparable: our study had a higher proportion of stage 1 patients (20%) and a lower proportion of patients with metastatic disease (30% in our series and 42% in the study of Meitar et al37 ), although the staging system was not the same (International Neuroblastoma Staging System v Pediatric Oncology Group); MYCN amplification was defined differently ( 10 copies in our study v three copies in Meitar’s study). Proportion of infants and unfavorable Shimada were similar in both series (52% and 30%, respectively, in our cohort v 44% and 28%, respectively, in Meitar’s study). The methodology used by these investigators was based on manual counting of vessels in the tumor by two reviewers: after hematoxylin and eosin staining, all vessels in a slide that contained primary tumor tissue were counted, tumoral area (excluding necrosis and calcification) in that slide was determined by an image processor, and a vascular index (total number of vessels per mm2) was calculated. In the study of Meitar et al,37 investigators determined a vascular index of 4.0 to be the cutoff point to distinguish low vascularity from high vascularity, but they did not explain the reason why this value was chosen instead of another. As discussed earlier, we preferred to choose a methodology that had been used previously50 in our laboratory with good results and little variability, as it was performed by a computerized analysis. For the statistical analysis, we decided to perform nonparametric tests, because we did not know whether our population had a normal distribution (66 patients only). Patients in whom angiogenesis was studied and the remaining patients from the N-II-92 study had the same clinical and biologic characteristics: as mentioned before, there was a significant number of infants (52%), although Spain did not have a national screening program for NB, which is known to increase the incidence of NB, especially in low stages.62,63 This high proportion of infants was parallel with a high proportion of "good" cases (stages 1, 2, and 4s). In concordance therewith, only one third of our tumors were considered to be unfavorable according to Shimada’s classification, and only 7% of the total expressed MYCN amplification. We did not find statistical differences in any of the vascular parameters according to age (infants and older children), in agreement with Meitar et al.37 Additionally, we did not observe statistical differences in vascular parameters when tumors were divided according to clinical stages (1, 2, 3, 4, and 4s) or Shimada’s classification (favorable v unfavorable). We studied TRK-A expression in 49 of these tumors. It could be expected that more differentiated tumors (strong TRK-A expression) would be less angiogenic, but this could not be demonstrated in our series. The three MYCN-amplified patients had mean values of their vascular parameters that did not differ from those of the MYCN-nonamplified patients, except for object number and volume, but the number of MYCN-amplified patients is too small to draw conclusions. It is widely accepted that angiogenesis is a basic process for local tumoral growth and metastases, but it is not the only one.64 By studying tumors at diagnosis and before treatment, we wanted to determine whether vascular parameters in the primary tumor could influence relapse occurrence and type of relapse. None of our samples was obtained from a patient in relapse or with progressive disease. We could not demonstrate that more angiogenic tumors are aggressive and more prone to metastasize, leading to more disseminated relapses. As far as local isolated relapse is concerned, we think this angiogenic process would not be important in our group of patients, because other factors could play a key role (persistent clones from the primitive tumor, changes in local immunity, and so on). These findings could have implications for therapy in different ways: although antiangiogenesis therapy could be theoretically efficacious in patients with NB, as Wassberg et al58 and Nagabuchi et al59 have demonstrated in experimental and murine models, it should be applied only in those patients with very angiogenic tumors (which is not the case in our series), and it should first be proven that this therapy does not harm healthy infantile organs and their growth. Those tumors that are less angiogenic and relapse locally would need a good local treatment (surgery, radiotherapy, [125I]metaiodobenzylguanidine), and systemic treatment would be secondary. OS and EFS for our group of patients is 75% and 55%, respectively, with a median follow-up of 54 months. When multivariate analysis was performed on more than 51 angiogenesis patients, enolase and Shimada were good predictors of survival. In conclusion, we could not demonstrate that vascular parameters influenced survival in this small group of patients from the N-II-92 study with a high number of favorable cases (52% infants, 35% with localized disease, only 7% with MYCN-amplified tumors, few events). Nonetheless, we consider that more objective and refined angiogenesis quantification methods are needed to include angiogenesis in prognostic studies in cancer and to measure response to future antiangiogenic therapy adequately. These objectives can only be achieved via large cooperative studies.
The following hospitals participated in the study: Hospital Infantil La Paz, Hospital Doce de Octubre, Hospital Niño Jesús, and Hospital Gregorio Marañón (Madrid); Hospital Infantil La Fe and Hospital Clínico (Valencia); Hospital Regional Carlos Haya (Málaga); Hospital San Joan de Deu and Hospital Santa Creu i San Pau (Barcelona); Hospital de Cruces (Vizcaya); Hospital General de Galicia (Santiago); Hospital Xeral (Vigo); Hospital Virgen del Camino (Pamplona); Hospital General and Hospital San Juan (Alicante); Hospital Central de Asturias (Oviedo); Hospital Torrecárdenas (Almería); Hospital Clínico Universitario (Tenerife); Hospital Miguel Servet (Zaragoza); Hospital Nen Jesus (Sabadell), Hospital Son Dureta (Palma de Mallorca); Hospital Teresa Herrera (La Coruña); Hospital Marqués de Valdecilla (Santander); Hospital Virgen de la Arrixaca (Murcia); Hospital General (Albacete); and Hospital Germans Trias i Pujol (Badalona, Spain).
Supported by grants no. FIS 98/0600 and FIS 94/1575. We thank Elisa Alonso and A. Sempere for technical support, and Drs J. Fernández and A. Verdeguer for critical review of the manuscript.
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Copyright © 2000 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
2 test to analyze contingency tables, Mann-Whitney U two-sample test and Kruskal-Wallis k-sample test (as the nonparametric alternatives to the t test and analysis of variance test), Kaplan-Meier survival curves and the log-rank test to compare survival curves, and the Cox proportional hazards model (forward, likelihood ratio test) to select prognostic factors in predicting survival. A P value less than .05 was considered statistically significant. 
