Background: Current staging systems for unresectable or metastatic neuroblastoma do not reliably predict responses to chemotherapy in infants under 1 year of age. Previous studies have indicated that the DNA content, or ploidy, of malignant neuroblasts can discriminate between good and poor responders in this group of patients, but the clinical utility of ploidy assessment has remained in question. Purpose: We tested, in a prospective nonrandomized study, the hypothesis that neuroblast ploidy could be used as the sole guide for treatment selection in infants with unresectable or metastatic tumors and could differentiate between those who would respond to our previous standard regimen and those who would benefit from an immediate switch to another therapy. Methods: One hundred seventy-seven infants were enrolled in this trial. Five of these infants were subsequently excluded (two ineligible, two lacking ploidy information, and one protocol violation); therefore, 172 patients were included in the study. One hundred thirty infants with hyperdiploid tumors (DNA index > 1.0; better prognosis in retrospective studies) were treated with a well-tolerated regimen of cyclophosphamide (150 mg/m 2 per day orally or intravenously ondays 1–7) and doxorubicin (35 mg/m 2 intravenously on day 8). Forty-two infants with diploid tumors (DNA index = 1.0; worse prognosis in retrospective studies) received cisplatin (90 mg/m 2 intravenously on day 1) and teniposide (100 mg/m 2 intravenously on day 3) after an initial course of cyclophosphamideplus doxorubicin. Statistical end points were response and long-term survival. In addition, we assessed within each ploidy group (i.e., patients with hyperdiploid tumors and those with diploid tumors) the prognostic significance of NMYC gene copy number, tumor stage, and other variables commonly measured in this disease. Results: Of the 127 assessable infants with hyperdiploid tumors, 115 (91%) had complete responses—85 after receiving five courses of cyclophosphamide plus doxorubicin and 30 after receiving further therapy including cisplatin plus teniposide. The 3-year survival estimate for the entire hyperdiploid group was 94% (95% confidence interval [CI] = 89%–98%). Nineteen (46%) of 41 assessable infants with diploid tumors were complete responders. The overall 3-year survival estimate for this group was 55% (95% CI = 39%–70%). Prognostic factor analysis indicated that NMYC gene amplification and an elevated serum lactate dehydrogenase level were statistically significant markers of higher risk disease within the diploid group (two-sided P values of .005 and .003, respectively). Only NMYC was predictive in the hyperdiploidgroup ( P = .003). Conclusion: Use of a prognostic staging system based on tumor cell ploidy, augmented with the NMYC gene copy number and serum level of lactate dehydrogenase,would very likely improve the treatment of infants with unresectable or metastatic neuroblastoma. Patients with diploid tumors characterized by an amplified NMYC locus represent a particularly unfavorable risk group that may benefit from innovative new therapies. [JNatl Cancer Inst 1997;89:373–80] The assessment of tumor cell biology and its relation to prognosis has been at the heart of efforts to improve therapy for the childhood cancers ( 1 , 2 ). Indirect measures of biologic heterogeneity-such as tumor size, primary site, degree of tumor progression,and histopathology-have all been used to devise staging systems and predict outcome, but none has provided a precise guide to clinical management (2–7). Observations made during the past decade indicate that genetic changes underlie the expression and maintenance of malignant phenotypes and are largely responsible for the extensive diversity of tumor cells ( 8 , 9 ). This suggests that tumor staging based on one or more genetic features could offer prognostic discrimination not available through unmodified clinico pathologic systems and thus could aid in the choice of the most appropriate therapy for in?dividual patients. Neuroblastoma arises from the adrenal medulla or other areas of the sympathetic nervous system and is the most common neoplasm affecting infants ( 7 ). Current staging systems for unresectable or metastatic neuroblastoma do not reliably predict responses to chemotherapy in infants, particularly among those under 1 year of age. In previous studies ( 10 , 11 ), we demonstrated the ability of tumor cell DNA content (ploidy) to predict responses to chemotherapy among infants with unresectable or metastatic neuroblastoma. Infants with hyperdiploid tumors consistently became long-term survivors after well-tolerated combination chemotherapy involving administration of cyclophosphamide plus doxorubicin, whereas the vast majority of those with diploid tumors had poor responses to this treatment. Thus, we postulated that neuroblast ploidy could be used prospectively to assign patients to standard therapy (as above) or to alternative regimens. In this nonrandomized study, cyclophosphamide plus doxorubicin was administered as primary chemotherapy to all infants with hyperdiploid tumors, while cisplatin plus teniposide was selected for those with diploid tumors on the basis of its promising activity against resistant neuroblastoma ( 12 ). We also assessed the prognostic role of NMYC gene amplification ( 11 , 13 ) and more commonly measured prognostic variables within ourploidy-based treatment systems. Infants under 1 year of age with previously untreated metastatic or unresectable neuroblastoma were eligible for Pediatric Oncology Group (POG) study 8743, in which patients were enrolled from May 1987 to October 1991. Minimum and maximum follow-up times were 18 days and 8.9 years, respectively (median, 5.6 years). The diagnosis of neuroblastoma was based on histologic examination of tumor specimens (or of bone marrow samples in the case of patients with increased catecholamine levels) and an abdominal or thoracic mass suggestive of neuroblastoma. All diagnoses were confirmed by the coordinating pathologist (V. Joshi). Post operative staging followed criteria of the Pediatric Oncology Group ( 14 ): stage A, localized resectable tumors; stage B, localized but unresectable tumors with negative noncontiguous lymph nodes; stage C, metastasis to noncontiguous lymph nodes; stage D, metastasis beyond the lymph nodes; and stage D s [stage IVS in the Childrens Cancer Group system ( 15 )], metastasis limited to liver, skin, and bone marrow. Infants with tumors determined to be of stage B, C, D, or D s were eligible for the study; those with stage A tumors received no therapy after surgical excision and were not enrolled. This investigation was approved by the institutional review boards of all participating Pediatric Oncology Group institutions ( see Appendix Table 1 ). Written informed consent was obtained from the parents or legal guardians of each child. Tumor or bone marrow samples containing greater than 10% malignant cells were shipped to St. Jude Children's Research Hospital and were used to determine the DNA content of neuroblasts; the DNA content then was converted to a DNA index (ratio of the modal G0 cell cycle phase/G1 cell cycle phase DNA content for neuroblastoma cells versus that of normal diploid cells) ( 10 , 11 ). A DNA index of approximately 1.0 corresponds to a diploid chromosome number; an index greater than 1.0 denotes hyperdiploidy. In patients with multiple stem lines, the line with the lowest DNA index was used for staging purposes. The heterogeneity of DNA content measurements within the hyperdiploid groups has not shown prognostic significance in neuroblastoma ( 10 ) and was therefore not considered a major source of response variability in this study. The lowest DNA index in a nondiploid tumor was 1.08. The NMYC gene copy number was determined by Southern blot analysis in tumor specimens submitted to the Washington University School of Medicine, St. Louis, MO, as described previously ( 11 , 13 , 16 ). Briefly, tumor DNA was digested, hybridized to the 2.0-kilobase pNb-1 probe for the NMYC oncogene ( 17 ), and radiolabeled ( 18 ). NMYC copy number was determined by serial dilution and laser densitometry. The gene was considered unamplified if the copy number was less than or equal to 3 per haploid genome and amplified if the number was greater than 3. Serum levels of lactate dehydrogenase (LDH) ( 19 ) and ferritin ( 20 ) were measured at participating institutions. A complete response was defined as a greater than 90% regression of tumor at the primary site with complete regression of all metastatic disease, including healing of bone lesions (i.e., decreased uptake of 99 technitium-diethylenetriaminepentaacetic acid or resolution of lytic lesions on plain roentgenograms). A partial response was considered to be a decrease greater than or equal to 50% but less than or equal to 90% in the volume of the primary tumor, witha greater than 50% decrease in the combined mass of all measurable metastatic lesions except bone lesions, no evidence of progression of any lesion, and no development of new disease. A partial response of bone metastases was defined as no increase in size or number of lesions by analysis of radiographic bone scanor plain roentgenograms. Minimum time to qualify as a response was 4 weeks. Patients not meeting the criteria for a complete or partial response were considered non responders, including those with a partial response at one site and progressive disease or no response at another site. One hundred seventy-seven infants were enrolled in this trial. Five of these infants were subsequently excluded (two in eligible, two lacking ploidy information, and one protocol violation). Thus, data are presented for 172 patients. During the interval from diagnosis to measurement of tumor cell ploidy, all patients were treated with a single course of cyclophosphamide (Cytoxan; Bristol-Myers Squibb, Princeton, NJ) at a dose of 150 mg/m 2 per day orally or intravenously for 7 days and doxorubicin (Adriamycin; Pharmacia, Dublin, OH) at a dose of 35 mg/m 2 intravenously on day 8, as previously described ( 10 , 11 , 21 ). They were then assigned to one of two treatment arms according tothe ploidy classification of their tumors. Hyperdiploid group. One hundred thirty infants with hyperdiploid tumors received four additional courses of cyclophosphamide plus doxorubicin at 21-day intervals. Those who developed progressive disease at either a primary or a metastatic site during this therapy were immediately switched to cisplatin (Platinol; Bristol-Myers Squibb) at a dose of 90 mg/m 2 intravenously on day 1 followed by teniposide at a dose of 100 mg/m 2 intravenously on day 3. Patients were eligible for second-look surgery if they had complete resolution of metastatic tumor in all sites (except the liver, where a partial response was acceptable) and if the primary tumor appeared to be at least 50% resectable. Those with grossly resected tumors and no evidence of residual disease were considered complete responders and had their therapy discontinued. Those with greater than 50% but less than gross total resections, evidence of active disease in non adherent lymph nodes, or residual neuroblastoma or ganglioneuroblastoma detected by liver biopsy received two additional courses of cisplatin plus teniposide, followed 21 days later by cyclophosphamide plus doxorubicin. Patients who were not eligible for second-look surgery were considered to have non responsive tumors and thus were switched to the cisplatin plus teniposide regimen. Diploid group. After their initial course of therapy with cyclophosphamide plus doxorubicin, 42 infants with diploid tumors were treated with cisplatin (90 mg/m 2 intravenously on day 1) and teniposide (100 mg/m 2 intravenously on day 3). Those with progressive disease at any time after the first two courses were declared treatment failures on cisplatin plus teniposide. Second-look surgery was performed after patients had received five courses of primary chemotherapy according to the same criteria that were applied to the hyperdiploid group. Patients who were in eligible for surgery received two additional courses of cisplatin plus teniposide and then were re-evaluated. Treatment was discontinued for patients with surgical evidence of a complete response; all other patients received three additional courses of cisplatin plus teniposide. Trimethoprim plus sulfamethoxazole prophylaxis on a 3-day-per-week schedule (150 mg/m 2 of trimethoprim orally divided twice a day) for prevention of Pneumocystis carinii pneumonitis was recommended for all patients. During treatment, each child was tested regularly for cardiac or renal dys-function and for other types of toxic effects. Follow-up surveillance included echocardiographic studies at 7 months, 1 year, and 2 years after the completion of therapy. Whenever possible, to measure ototoxicity, brain-stem audiometry was conducted at these intervals in children treated with cisplatin plus teniposide. Physical examinations were performed every 2–6 months during the first 3 years after treatment and annually thereafter. Definitions of toxicity were as described in the Common Toxicity Criteria (Regulatory Affairs Branch, Cancer Therapy Evaluation Program, Division of Cancer Treatment, Diagnosis, and Centers, National Cancer Institute, Bethesda, MD). Major considerations in planning the study were ( a ) the very high rate of complete responses (approximately 90%) among patients with hyperdiploid tumors treated with cyclophosphamide plus doxorubicin in the past, contrasted with the very low rate among patients with diploid tumors receiving the same combination (one responder among 15 infants treated) ( 11 ); ( b ) the ineffectiveness of late administration of alternative therapy in producing long-term survival in patients with disease resistant to cyclophosphamide plus doxorubicin ( 11 ); and ( c ) the expected slow accrual of patients to the diploid group. Thus, we chose a nonrandomized study design to test our central hypothesis that tumor cell ploidy measurements identify two distinct groups of patients, one (hyperdiploid) that to cyclophosphamide plus doxorubicin therapy and another that is to respond to this therapy and an immediate switch to an alternative The primary end points in this study were the response to chemotherapy and overall as measured from the of were as a of time by the of and ( ). between and survival were determined with the the ( ). This on which can be the number of is The and confidence intervals for NMYC gene serum and primary site in patients with diploid tumors were by the recommended by ( , ). between ploidy classification and features were tested with A P of was to indicate Of the 172 infants treated in this had hyperdiploid tumors and 42 had diploid tumors. was a between these groups in the of tumor and other features ( Table 1 ). the majority of patients with diploid tumors had stage D disease, those in the hyperdiploid group were more likely to have stage or disease. adrenal tumors, which have been with an unfavorable prognosis ( ), were more common among patients with diploid tumors than among patients with hyperdiploid tumors versus P = The NMYC copy number in was to be amplified in 10 patients with diploid tumors with patients with hyperdiploid tumors versus P Diploid tumors were more in ( P = and in patients with elevated levels of serum ( P responses were in 115 (91%) of the 127 assessable infants with hyperdiploid tumors of the patients were not one of a late protocol and two of of these patients, the complete response from treatment involving five courses of cyclophosphamide plus doxorubicin. a complete response in the other 30 patients, either cisplatin plus teniposide therapy to respond to the first = or courses of cisplatin plus teniposide with cyclophosphamide plus doxorubicin = were of the NMYC gene was detected in patients with hyperdiploid tumors, two of were considered resistant to primary treatment. Of the 41 assessable infants in the diploid group (one of the 42 patients was not assessable of to therapy through the 19 (46%) had complete responses to cisplatin plus teniposide. among patients with diploid tumors to be by the NMYC copy All 172 infants were assessable for survival. 3 years of an 94% (95% confidence interval [CI] = of the patients with hyperdiploid tumors were ( 1 ). Of the 115 patients as complete responders and the five as partial responders who all more than 2 years after of the infants in the hyperdiploid group with amplified NMYC of tumor at and the other three patients are and in complete at and years, after the initial The 3-year survival estimate for the diploid group was 55% (95% CI = 39%–70%). Of the 30 from in patients with NMYC gene 10 other were with a non amplified NMYC and were with an NMYC of between patients with or amplified NMYC indicated survival in the ( 2 Only five of the 13 patients with diploid tumors and a partial response were at the time of after bone marrow and two after one has a cell survival for infants with unresectable or metastatic neuroblastoma treated according to ploidy group. The patients with hyperdiploid tumors and NMYC gene amplification are by and the 10 patients with diploid tumors and NMYC gene amplification are by Patients at risk of at the of each interval are indicated the A major of the study was to identify clinical or biologic risk that the predictive of neuroblast patients with hyperdiploid tumors, NMYC gene amplification prognostic significance in a analysis ( Table 2 ); further study was not of the in the hyperdiploid group ( ). tumor site, NMYC copy number, and serum as significant of in the analysis for patients with diploid tumors ( Table 2 ). The prognostic of primary site became ( P was made for serum level and was made for NMYC gene amplification ( Table 3 ); of the features significance the analysis was by site ( P = .005 and respectively). NMYC gene amplification serum level after for each these features were not in as as four of the 20 witha diploid in children who no amplification of NMYC and an elevated serum Thus, the a of patients at higher risk disease would not be by NMYC copy number survival for infants with diploid with or amplified NMYC Patients at risk of at the of each interval are indicated the was commonly in patients on treatment but higher of patients with hyperdiploid tumors who had been treated on the cyclophosphamide plus doxorubicin regimen were to the for ( Table 4 ). with the two were for the diploid group versus for the hyperdiploid infants with diploid tumors had a decrease in after receiving cisplatin of mg/m 2 , which of this treatment. infants with hyperdiploid tumors had a 50% decrease in after receiving mg/m 2 of cisplatin as maintenance therapy. The of with cisplatin plus teniposide therapy could not be determined of the in test in patients developed after receiving cisplatin of and mg/m 2 and infants who had received doxorubicin of mg/m 2 and mg/m 2 , 3 in as determined and were treated with developed carinii pneumonitis while they were receiving cyclophosphamide plus doxorubicin therapy trimethoprim plus sulfamethoxazole was a single of in a child on the cyclophosphamide plus doxorubicin regimen. and disease stage at diagnosis have been the most for in their selection of for children with neuroblastoma. In complete surgical of localized tumors further treatment rate of ( 14 ), whereas for patients with or disease, the is less the child with disease is than 2 of is of the treatment used ( 11 , ). infants than 1 year have long-term survival limited or whereas treated with the same initial or after ( 10 , 11 , ). We have that genetic for of the prognosis of neuroblastoma in infants ( 10 , 11 ) therefore, could be used to guide the choice of primary treatment. study to our the first prospective test of this The that a staging system based on tumor cell ploidy combined with NMYC copy number, serum and other biologic markers ( , 30 ) would very likely improve the treatment selection for infants with unresectable or metastatic neuroblastoma. Of the infants with hyperdiploid tumors, 10 have after treatment with cyclophosphamide plus doxorubicin, administered or followed by cisplatin plus teniposide survival CI 4 89%–98%). This prospectively made on a of infants in an Pediatric Oncology Group study ( 11 ). therapy with cyclophosphamide plus doxorubicin in of patients with hyperdiploid tumors, this therapy effects. of the patients with NMYC gene amplification in the hyperdiploid group complete and three are of disease. Thus, of the NMYC oncogene to be a poor prognostic in hyperdiploid that be used to identify the who is likely to on the primary regimen. In an study ( ), high serum levels were predictive of a poor In the analysis performed in the study, high serum levels were suggestive of a poor outcome, but the was not statistically significant ( P = the other variables including disease stage, not in risk within this group. The biologic for the increased of hyperdiploid neuroblastoma in infants One by one in study of hyperdiploid cells ( ), would be a to in the of as that by is by the high levels of factor by hyperdiploid tumors; this factor may have an role in neuroblastoma regression or ( ). of the patients with diploid tumors to cisplatin plus teniposide after an initial cyclophosphamide plus the survival estimate for the entire diploid group was 55% (95% CI = 39%–70%). ( 10 , 11 ), we would have a low complete response rate (approximately had the cyclophosphamide plus doxorubicin combination been used and a survival rate of less than with of therapy after disease progression ( 11 ). NMYC gene amplification not predict initial to chemotherapy in these patients, was with in with retrospective ( , ). Only one of the 10 infants who had amplified NMYC gene was a at the time of with 14 of 20 who had diploid tumors and NMYC of our confirmed the of NMYC gene amplification as a of on the cisplatin plus teniposide regimen. approximately one of patients in the diploid in this study who not have an amplified NMYC locus have also be to this genetic with other markers to a staging system that has a more predictive A from of chromosome ( 30 ) and increased levels of ( ) have as of disease in patients NMYC gene these genetic features the features in our study prospective in infants with neuroblastoma. have that stage D s neuroblastoma is in a tumor that therapy ( 35 ). In our tumors have the same as other in We would within ploidy the clinical for stage tumors was not from that for tumors of other ( Table 2 ). Thus, we do not a management for patients with tumors in this but that they be treated according to ploidy and NMYC copy cisplatin plus teniposide in the with diploid tumors who NMYC gene is a to therapy for these patients. ( ) at the Cancer an in infants with stage [stage D in the Pediatric Oncology Group system ( 15 neuroblastoma treated with a regimen that included doxorubicin, and ploidy NMYC were that the of this regimen in the treatment of patients with disease A is the and that can from long-term administration of cisplatin to improve clinical in neuroblastoma patients, the Pediatric Oncology Group is with in children with disease ( , ), ( ), and ( and is on factor to decrease toxicity and to more of these improve in infants with diploid tumors and NMYC gene amplification the prognosis for this to be we would to these as of neuroblastoma in which has all combination chemotherapy ( ). Thus, of the innovative in children 1 year of chemotherapy with stem cell ( ), ( ), and gene therapy ( 41 for this very of the disease.
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