In the last century, the progressive introduction of active immunization

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1 635 Assessment of Humoral Immunity to Poliomyelitis, Tetanus, Hepatitis B, Measles, Rubella, and Mumps in Children after Chemotherapy Matteo Zignol, M.D. 1 Marta Peracchi, M.D. 2 Gloria Tridello, Ph.D. 3 Marta Pillon, M.D. 3 Federica Fregonese, M.D. 1 Ruggiero D Elia, M.D. 1 Luigi Zanesco, M.D. 3 Simone Cesaro, M.D. 3 1 Department of Pediatrics, Faculty of Medicine and Surgery, University of Padua, Padua, Italy. 2 Institute of Microbiology and Virology, University of Padua, Padua, Italy. 3 Pediatric Hematology and Oncology Clinic, University of Padua, Padua, Italy. BACKGROUND. To evaluate the effect of chemotherapy on humoral immunity to vaccine-preventable disease, the authors investigated the persistence of protective antibody titers in a group of who were alive and well after they were treated for pediatric malignancies. METHODS. Serum antibody levels were evaluated for polio, tetanus, hepatitis B, rubella, mumps, and measles in 192 children. The terms lack of immunity and loss of immunity, respectively, were used to describe the absence of immunity in who were tested only after chemotherapy and in who were tested both before and after chemotherapy and determined to have immunity before chemotherapy. RESULTS. Overall, the absence of a protective serum antibody titer for hepatitis B, measles, mumps, rubella, tetanus, and polio was detected in 46%, 25%, 26%, 24%, 14%, and 7% of, respectively. On univariate analysis, loss of antibodies against rubella, mumps, and tetanus was associated significantly with younger age (P 0.001, P 0.02, and P 0.001, respectively), and loss of antibodies against measles was significantly associated with younger age and female gender (P and P 0.008, respectively). The administration of 59 booster vaccinations to 51 who had lost 1 protective antibody titer resulted in an overall response rate of 93%. CONCLUSIONS. Chemotherapy induced different rates of loss of protective antibody titers depending on the type of vaccination administered. This finding may be responsible for the failure of vaccination programs for who have undergone chemotherapy. The administration of a booster dose after the completion of chemotherapy is a simple and cost-effective way to restore humoral immunity against most vaccine-preventable diseases. Cancer 2004;101: American Cancer Society. The authors thank Stefania Gallo for her skilful editorial assistance. Address for reprints: Simone Cesaro, M.D., Pediatric Hematology and Oncology Clinic, University of Padua, Via Giustiniani 3, Padova, Italy; Fax: (011) ; simone. cesaro@unipd.it Received February 2, 2004; revision received April 2, 2004; accepted April 26, KEYWORDS: pediatric malignancy, chemotherapy, vaccination, tetanus, poliomyelitis, measles, rubella, mumps, hepatitis B. In the last century, the progressive introduction of active immunization by vaccination has played a fundamental role in preventing several infectious diseases, such as polio, tetanus, diphtheria, and (more recently) hepatitis B, mumps, rubella, and measles. 1,2 Today, vaccination still represents a key public health tool for reducing infectious morbidity and mortality all over the world. 3,4 For the time being, chemotherapy and radiotherapy are the only curative treatments for children who are affected by malignancy, and their use in recent decades has led to significant improvements in disease-free survival for with all types of tumors. 5 An unavoidable drawback of chemotherapy is hematologic toxicity, which results in tran American Cancer Society DOI /cncr Published online 18 June 2004 in Wiley InterScience (

2 636 CANCER August 1, 2004 / Volume 101 / Number 3 sient immunodeficiency that lasts for up to 6 12 months after the end of the treatment. 6,7 The younger the patient is, the longer immunodepression lasts, 8 and B lymphocytes are particularly susceptible to this effect. 9,10 In who have undergone hematopoietic stem cell transplantation (HSCT), protective antibody vaccination titers are compromised seriously and even definitively, and international guidelines recommend revaccination from 6 months to months after HSCT There few published reports, however, on which to base recommendations for who have undergone chemotherapy for leukemia or solid tumors, and this issue remains a matter of investigation and discussion In particular, it has yet to be established whether the degree of immune dysfunction after standard chemotherapy demands a booster dose outside of the recommended schedule, revaccination, or no intervention at all. To evaluate the effect of chemotherapy on humoral immunity to vaccine-preventable diseases, we investigated the persistence of protective antibody titers in a group of who were alive and well after treatment for pediatric malignancies. Immune responses to booster doses and revaccinations after chemotherapy also were assessed. MATERIALS AND METHODS One hundred ninety-two who had been treated successfully for pediatric malignancies were evaluated after chemotherapy for humoral immunity against vaccine-preventable diseases (e.g., hepatitis B virus [HBV], tetanus, polio, measles, mumps, and rubella). All had been diagnosed at the Pediatric Hematology Oncology Clinic at the University of Padua (Padua, Italy) between January 1993 and December 1998; these had completed first-line chemotherapy and were survivors with disease that was in complete remission. All were screened for human immunodeficiency virus. Patients who had developed recurrent disease and/or had undergone HSCT (autologous or allogeneic) and children age 1 year who had been diagnosed with a malignancy before completing the compulsory vaccination schedule were excluded from the study. The Italian vaccination program involves compulsory vaccinations for tetanus, polio, diphtheria, and hepatitis B, in addition to other strongly recommended vaccinations (e.g., vaccinations against measles, mumps, rubella, pertussis, and Haemophilus influentiae). Primary vaccination schedules for tetanus, polio, diphtheria (and pertussis, if requested by a parent), and HBV included 3 doses of vaccine administered at 4 months, 6 months, and 11 months after birth, respectively; in contrast, measlesmumps-rubella vaccinations are administered in a single dose at age months. Our policy is to suspend any vaccination schedule upon diagnosis of a malignancy (and especially vaccination schedules involving attenuated viral agents) until at least 12 months after the end of chemotherapy. The patient s parents and pediatrician are given no recommendations concerning the routine administration of vaccination booster doses or revaccinations 6 12 months after the end of the treatment. The current study included 114 males and 78 females with a median age at diagnosis of 6 years (range, 1 18 years). One hundred thirty-five (70.3%) had hematologic malignancies, including acute lymphoblastic leukemia (ALL) in 85, Hodgkin lymphoma in 25, non-hodgkin lymphoma in 19, acute myeloid leukemia in 3, and multisite/multifocal Langerhans cell hystiocytosis in 3. Fifty-seven (29.7%) had solid tumors, including Wilms tumor in 18, soft tissue sarcoma/rhabdomyosarcoma in 17, neuroblastoma in 5, central nervous system tumors in 6, hepatoblastoma in 1 patient, osteosarcoma in 3, teratoma in 1 patient, and other malignancies in 6. Treatment was administered according to the cooperative protocols of the Italian Pediatric Hematology Oncology Association, and details on the chemotherapy administered and on patient outcomes have been reported elsewhere Data on previous vaccinations and childhood infectious diseases were collected from the hospital s clinical files and were confirmed by vaccination certificates. All had completed the primary vaccination schedule for tetanus, polio, and HBV; whereas immunity to measles, mumps, and rubella was acquired either through previous clinical infection or through vaccination. Serum antibody titers for tetanus, polio, diphtheria, hepatitis B, measles, mumps, and rubella were determined routinely at first admission (usually on the first or second day) before any hemocomponent transfusion, unless the patient s low weight, severe anemia, or compromised clinical condition prevented sufficient blood sampling for serology tests. Posttreatment serologic quantities were assessed only for the purposes of the study. The median interval between chemotherapy and antibody serum titer assessment was 15 months (range, months). All were tested at the first follow-up visit at our center and at least 6 months after the last hemocomponent transfusion. All serologic tests were conducted once using fresh serum samples before and/or after chemotherapy, and no serum was stored. Consequently, the total numbers of assessed for HBV, measles,

3 Vaccination Antibodies after Chemotherapy/Zignol et al. 637 TABLE 1 Rates of Lack of Protective Serum Antibody Titers for Hepatitis B, Measles, Mumps, Rubella, Tetanus, and Polio in Patients after Chemotherapy for Pediatric Malignancies No. of Hematologic malignancies (%) Solid tumors (%) Total (%) HBV /73 (44) 21/43 (49) 53/116 (46) Measles /98 (23) 12/40 (30) 35/138 (25) Mumps /87 (22) 16/40 (40) 35/127 (28) Rubella /90 (22) 11/41 (27) 31/131 (24) Tetanus /116 (15) 5/46 (11) 22/162 (14) Polio 137 7/97 (7) 3/40 (8) 10/137 (7) HBV: hepatitis B virus. mumps, rubella, tetanus, and polio differ due to insufficient blood samples or the temporary unavailability of testing kits for some. To make the results clearer, we used the term lack of immunity to describe the absence of immunity in who were tested only after chemotherapy and the term loss of immunity to describe the absence of immunity in who were tested both before and after chemotherapy and were determined to have immunity before chemotherapy. Informed consent was obtained from all or from their parents. Antibody Determination Commercial kits were used to determine antibody levels according to the manufacturer s instructions. Antibody levels against tetanus toxoid were assessed using the passive hemoagglutination test (Tetan Test; Sieroterapico Soc. Coop., Milan, Italy). Titers 1:64 were assumed to be indicative of protective levels. Antibody levels against poliomyelitis were evaluated using a complement fixation test for polioviruses 1, 2, and 3 (Virion Ltd., Ruschlikon, Switzerland), with protection indicated by titers 1:2. Levels of antibodies against hepatitis B surface antigen, measles, mumps, and rubella were assessed using a standard automated immunoassay processor (Behring Elisa Processor III; Dade Behring, Marburg, Germany) of the following immunoenzymatic tests (Dade Behring): Enzygnost anti-hbs II (cutoff level, 10 international units [IU] per liter); Enzygnost antimeasles virus/immunoglobulin G (IgG); Enzygnost antimumps virus/igg; and Enzygnost antirubella virus/igg. The cutoff level for the rubella assay was 15 IU/L, whereas qualitative assays were used for measles and mumps. Each assay was performed with positive and negative controls, and internal quality controls were used in in vitro assay procedures. Assessment of Response to Vaccination after Chemotherapy A single dose of vaccine (booster shot) for one or more vaccinations was offered to who had a loss of a measurable antibody titers according to the evaluations performed before and after chemotherapy. Antibody responses were evaluated after 4 weeks. The vaccines used were as follows: tetanus toxoid (Imovax Tetano; Aventis Pasteur MSD, Rome, Italy), inactivated polioviruses (Poliovax; Chiron, Siena, Italy), recombinant HBV protein (Hbvaxpro; Aventis Pasteur MSD), live attenuated measles virus (Morbilvax; Chiron), live attenuated rubella virus (Rudivax; Aventis Pasteur MSD), and live attenuated mumps virus (Vaxipar; Chiron). Statistical Methods Data were entered into a database for analysis. Chisquare tests or Fisher exact tests were used to compare proportions, as appropriate, and the Mann Whitney test was used to compare medians of continuous variables. The role of gender (male vs. female), age at diagnosis (less than or greater than the median age), and underlying disease (leukemia and lymphoma vs. solid tumors) in predicting the loss of protective antibody levels was explored on univariate analysis, which was limited to who had their serum antibody titers determined both before and after chemotherapy. A P value of 0.05 was considered significant. Factors with P values 0.1 on univariate analysis were entered into a logistic stepwise regression model, with adjustment of covariates for loss of vaccine antibodies. The SAS software package (SAS Institute, Cary, NC) was used for all analyses. RESULTS Data on the lack of protective serum antibodies in who had undergone chemotherapy for pedi-

4 638 CANCER August 1, 2004 / Volume 101 / Number 3 TABLE 2 Rates of Loss of Protective Serum Antibody Titers in Patients Tested for Immunity before and after Chemotherapy No. of Hematologic malignancies (%) Solid tumors (%) Total (%) HBV 67 27/52 (52) 8/15 (53) 35/67 (52) Measles 92 20/78 (26) 3/14 (21) 23/92 (25) Mumps 77 12/65 (18) 4/12 (33) 16/77 (21) Rubella 76 14/67 (21) 0/9 (0) 14/76 (18) Tetanus /86 (13) 2/16 (13) 13/102 (13) Polio 77 6/66 (9) 0/11 (0) 6/77 (8) HBV: hepatitis B virus. atric malignancies are presented in Table 1. These data differed depending on the infectious disease and the type of malignancy, with a lack of HBV immunity observed in 46% of, a lack of measles immunity observed in 25% of, a lack of mumps immunity observed in 28% of, a lack of rubella immunity observed in 27% of, a lack of tetanus immunity observed in 14% of, and a lack of polio immunity observed in 7% of. Table 2 summarizes the absence of serum protective antibodies in who were tested both before and after chemotherapy, with losses of HBV immunity observed in 52% of, losses of measles immunity observed in 25% of, losses of mumps immunity in 21% of, losses of rubella immunity in 18% of, losses of tetanus immunity in 13% of, and losses of polio immunity in 8% of. Stratifying the latter group of according to previous immunization method (clinical infection vs. vaccination), rates of loss of antibodies against measles, mumps, and rubella were 0%, 11%, and 0% versus 27%, 17%, and 17%, respectively. Due to the limited cohort size, the chi-square test was performed only for measles and yielded a significance level of P Among who lost 1 antibody type, no statistically significant difference emerged with respect to the interval between the end of chemotherapy and serologic evaluation, with an interval of 6 months for 6 of 24 (25%), an interval of 6 and 12 months for 13 of 24 (54%), and an interval of 12 months for 5 of 24 (21%) (P 0.9). Fifty-nine booster vaccinations were administered to 51 who had lost 1 protective vaccination serum titer. Table 3 summarizes the results of these booster vaccinations; all but 4 booster vaccinations (which were administered to a total of 3 ) resulted in protective antibody serum titers, for an TABLE 3 Recovery of Protective Antibody Titers after Booster Vaccination in Patients Who Lost Humoral Immunity, According to Serum Tests before and after Chemotherapy No. of Hematologic malignancies Solid tumors Total (%) HBV 32 22/23 7/9 29/32 (91) Measles 5 4/5 4/5 (80) Mumps 1 1/1 1/1 (100) Rubella 5 5/5 5/5 (100) Tetanus 10 8/8 2/2 10/10 (100) Polio 6 5/5 1/1 6/6 (100) HBV: hepatitis B virus. overall efficacy of 93% (55 of 59 ). Quantitative determination of the increases in serum titers was performed for 13 of 29 who received a booster dose of HBV; the median serum titer 4 weeks after booster administration was 123 IU/L (range, IU/L). The roles of gender (male vs. female), age at diagnosis (less than or greater than the median age), and underlying disease (leukemia and lymphoma vs. solid tumors) in the loss of protective serum antibody levels for who were tested before and after chemotherapy were explored on univariate analysis (Table 4). The only predictive factor for loss of immunity against mumps, rubella, and tetanus was younger age (P 0.021, P 0.001, and P 0.01, respectively). The predictive factors for loss of immunity against measles were gender (P 0.008) and age (P ). On multivariate analysis, the significance of younger age and gender in predicting the loss of measles immunity was confirmed (younger age: P 0.002; relative risk, 7.1; 95% confidence interval, ; gender: P 0.02; relative risk, 3.6; 95% confidence interval, ).

5 Vaccination Antibodies after Chemotherapy/Zignol et al. 639 TABLE 4 Univariate Analysis of Factors Associated with the Loss of Protective Antibody Titers M vs. F P value Age (yrs) P value Underlying disease a P value Total no. of HBV 23/38 vs. 12/ vs /52 vs. 8/ Measles 7/50 vs. 16/ vs /78 vs. 3/ Mumps 7/44 vs. 9/ vs /65 vs. 4/ Rubella 6/44 vs. 8/ vs /67 vs. 0/ Tetanus 5/61 vs. 8/ vs /86 vs. 2/ Polio 4/45 vs. 2/ vs /66 vs. 0/ M: male: F: female; HBV: hepatitis B virus. a Leukemia/lymphoma versus solid tumor. DISCUSSION The current study demonstrated that pediatric who have undergone chemotherapy are prone to lose the protective serum antibody titers acquired from previous vaccinations. The loss of protective immune response varied according to the type of vaccination. Although treatment strategies for with leukemia usually differ from treatment strategies for with solid tumors in terms of duration (2 years vs. 1 year), schedule (continuous vs. cyclic), and type of agents used (with steroids being heavily used in leukemia treatment protocols), the underlying disease did not significantly affect the percentages of who lost protective antibody titers; in contrast, younger age significantly affected the chemotherapy-induced loss of antibodies against measles, mumps, and rubella. The reason for the loss of antibodies acquired on vaccination is not fully understood, but such losses have been linked to more severe susceptibility to chemotherapy and to longer B lymphocyte recovery times. Caver et al. observed profound B-cell lymphopenia in 16 who were receiving maintenance treatment for ALL lymphopenia that was unpredictable based on total white blood cell and lymphocyte counts. 9 Significant decreases in intramedullary plasma cell counts also have been observed during chemotherapy for ALL. 18 Mustafa et al. demonstrated that children with malignancies had specific and nonspecific immune alterations 9 12 months after the end of chemotherapy and that younger appeared to have more persistent alterations; in the latter group, antibody responses to immunization still were defective 12 months after the end of the therapy. 8 With overall losses of protective serum antibody titers in 8% and 13% of, respectively, immunity against polio and immunity against tetanus were least affected by chemotherapy. It has been demonstrated that in adults, chemotherapy has a stronger effect on humoral vaccination acquired immunity. Hamarstrom et al. evaluated antitetanus immune responses after chemotherapy/ radiotherapy in 206 (28% of whom were age 20 years); loss of specific immunity was detected in 36% of who were treated for acute myeloid leukemia, 56% of who were treated for ALL, 18% of who were treated for chronic myeloid leukemia, 54% of who were treated for lymphoma, and 31% of who were treated for myeloma. In that study, it is noteworthy that the loss of antibody response was more frequent and more severe in older compared with younger. 19 Italian policy regarding born before 1998 was to administer booster doses for polio and tetanus at age 5 6 years and to administer a booster dose for tetanus at age 10 years. This policy may help to explain the role of younger age in determining the loss of humoral immunity against tetanus. However, humoral immunity against polio was not influenced by age. Chemotherapy induced a loss of protective serum antibody titers for rubella, mumps, and measles in 18%, 21%, and 25% of, respectively. These findings are consistent with other published data. Feldman et al. evaluated a group of 39 children who were treated for leukemia (and who had been vaccinated previously for measles, mumps, and rubella) at diagnosis and at the end of chemotherapy. Those authors found decreases in seropositivity rates of 13% for measles, 18% for mumps, and 21% for rubella. 21 Nilsson et al. analyzed serum antibody levels in 43 children after chemotherapy and demonstrated the persistence of protective levels against measles and rubella in only 60% and 72% of, respectively. 18 Immunity against HBV was most significantly affected by chemotherapy in the current study. The loss of protective antibody serum titers was recorded in

6 640 CANCER August 1, 2004 / Volume 101 / Number 3 52% of who were evaluated before and after chemotherapy. This finding suggests that after chemotherapy, such may constitute a subpopulation that is susceptible to HBV infection in areas in which extensive programs of HBV vaccination for the overall pediatric population have been undertaken. In the current study, the administration of a combined total of 59 booster vaccinations for polio, tetanus, and HBV enabled all but 3 to recover protective antibody serum titers, suggesting that chemotherapy did not entirely abrogate the specific humoral memory in with undetectable serum antibody titers after chemotherapy. Thus, the administration of booster doses, rather than the selection of to revaccinate on the basis of serologic screening after chemotherapy, may be a simple, costeffective, and useful measure for restoring immunity in with malignant disease. Moreover, revaccination for rubella, mumps, and measles restored protective serum antibody titers in almost all, confirming that the impaired capacity to mount an immune response is temporary and that this capacity tends to return to normal 1 year after the end of chemotherapy. In conclusion, the current study demonstrates that chemotherapy leads to different rates of loss of protective serum antibody titers for different types of infectious disease. This finding may account for the failure of vaccination programs for who have undergone chemotherapy. Administration of a booster dose 12 months after the completion of chemotherapy is a simple and cost-effective way to restore humoral immunity against most vaccine-preventable diseases. REFERENCES 1. Centers for Control and Prevention. Ten great public health achievements United States, MMWR Morb Mortal Wkly Rep. 1999;48: Centers for Control and Prevention. Summary of notifiable diseases United States, MMWR Morb Mortal Wkly Rep. 2002;49: Centers for Control and Prevention. Recommended childhood immunization schedule United States, MMWR Morb Mortal Wkly Rep. 2002;51: Peter G. Immunization practices. In: Berman RE, Kliegman RM, Jenson HB, editors. Nelson textbook of pediatrics (17th edition). Philadelphia: W.B. Saunders, 2003: Smith MA, Ries LA. Childhood cancer: incidence, survival, and mortality. In: Pizzo PA, Poplack DG, editors. Principles and practice of pediatric oncology (4th edition). Philadelphia: Lippincott-Williams and Wilkins, 2002: Alanko S, Pelliniemi TT, Salmi TT. Recovery of blood B- lymphocytes and serum immunoglobulins after chemotherapy for childhood acute lymphoblastic leukemia. Cancer. 1992;69: Alanko S, Salmi TT, Pelliniemi TT. Recovery of blood T-cell subsets after chemotherapy for childhood acute lymphoblastic leukemia. Pediatr Hematol Oncol. 1994;11: Mustafa MM, Buchanan GR, Winick NJ, et al. Immune recovery in children with malignancy after cessation of chemotherapy. J Pediatr Hematol Oncol. 1998;20: Caver TE, Slobod KS, Flynn PM, et al. Profound abnormality of the B/T lymphocyte ratio during chemotherapy for pediatric acute lymphoblastic leukemia. Leukemia. 1998;12: Ito C, Ewans WE, McNinch L, et al. Comparative cytotoxicity of dexamathasone and prednisone in childhood lymphoblastic leukemia. J Clin Oncol. 1996;14: Ljungman P, Wiklund-Hammarsten M, Duraj V, et al. Response to tetanus toxoid immunization after allogeneic bone marrow transplantation. J Infect Dis. 1990;162: Ljungman P, Lewensohn-Fuchs I, Hammarstrom V, et al. Long term immunity to measles, rubella and mumps in after allogeneic bone marrow transplantation. Blood. 1994;84: Hammarstrom V, Pauksen K, Bjorkstrand B, Simonsson B, Oberg G, Ljungman P. Tetanus immunity in autologous bone marrow and blood stem cell transplantation recipients. Bone Marrow Transplant. 1998;22: Ljungman P. Immunization of transplant recipients. Bone Marrow Transplant. 1999;23: Singhal S, Mehta J. Reimmunization after blood or marrow stem cell transplantation. Bone Marrow Transplant. 1999;23: Ljungman P, Cordonnier C, de Bock R, et al. Immunizations after bone marrow transplantation: results of a European survey and recommendations from the Infectious s Working Party of the European Group for Blood and Marrow Transplantation. Bone Marrow Transplant. 1995;15: Avigan D, Pirofski LA, Lazarus HM. Vaccination against infectious disease following hematopoietic stem cell transplantation. Biol Blood Marrow Transplant. 2001;7: Nilsson A, De Milito A, Engstrom P, et al. Current chemotherapy protocols for childhood acute lymphoblastic leukemia induce loss of humoral immunity to viral vaccination antigens. Pediatrics. 2002;109:e Hamarstrom V, Pauksen K, Svensson H, Öberg G, Paul C, Ljungman P. Tetanus immunity in with hematological malignancies. Support Care Cancer. 1998;6: Van den Does-Van der Berg A, Hermans J, Nagel J, van Steenis G. Immunity to diphtheria, pertussis, tetanus and poliomyelitis in children with acute lymphoblastic leukemia after cessation of therapy. Pediatrics. 1981;67: Feldman S, Andrew M, Norris M, McIntyre B, Lyer R. Decline in rates of seropositivity for measles, mumps, and rubella antibodies among previously immunized children treated for acute leukemia. Clin Infect Dis. 1998;27: Conter V, Arico M, Valsecchi MG, et al. Long-term results of the Italian Association of Pediatric Hematology and Oncology (AIEOP) acute lymphoblastic leukemia studies, Leukemia. 2000;14: Conter V, Arico M, Valsecchi MG, et al. Intensive BFM chemotherapy for childhood ALL: interim analysis of the AIEOP-ALL91 study. Associazione Italiana Ematologia Oncologia Pediatrica. Haematologica. 1998;83:

7 Vaccination Antibodies after Chemotherapy/Zignol et al Amadori S, Testi AM, Arico M, et al. Prospective comparative study of bone marrow transplantation and postremission chemotherapy for childhood acute myelogenous leukemia. The Associazione Italiana Ematologia ed Oncologia Pediatrica Cooperative Group. J Clin Oncol. 1993;11: De Bernardi B, Nicolas B, Boni L, et al. Disseminated neuroblastoma in children older than one year at diagnosis: comparable results with three consecutive high-dose protocols adopted by the Italian Co-Operative Group for Neuroblastoma. J Clin Oncol. 2003;21: Cecchetto G, Bisogno G, Treuner J, et al. Role of surgery for nonmetastatic abdominal rhabdomyosarcomas: a report from the Italian and German Soft Tissue Cooperative Groups studies. Cancer. 2003;97: Cecchetto G, Carli M, Sotti G, et al. Importance of local treatment in pediatric soft tissue sarcomas with microscopic residual after primary surgery: results of the Italian Cooperative Study RMS-88. Med Pediatr Oncol. 2000;34: Vecchi V, Pileri S, Burnelli R, et al. Treatment of pediatric Hodgkin disease tailored to stage, mediastinal mass, and age. An Italian (AIEOP) multicenter study on 215. Cancer. 1993;72: Rosolen R, Pillon M, D Amore E, et al. Analysis of the AIEOP LNH-92 protocol for the diagnosis and treatment of pediatric non-b cell non-hodgkin s lymphomas. J Pediatr Hematol Oncol. 2003;25: Pillon M, Burnelli R, Cesaro S, et al. Results of the AIEOP NHL-92 protocol for the treatment of pediatric anaplastic large cell lymphomas. J Pediatr Hematol Oncol. 2003; 25:58.