Altered spectrin dimer-dimer association and instability of erythrocyte membrane skeletons in hereditary pyropoikilocytosis.

Hereditary pyropoikilocytosis (HPP) is a hemolytic anemia characterized by microspherocytosis, poikilocytosis, and an unusual thermal sensitivity of erythrocytes. We have investigated the contribution of abnormal membrane skeletal assembly to these abnormal HPP erythrocyte properties. Skeletons prepared from fresh HPP ghosts with Triton X-100 were considerably more fragile than skeletons from control erythrocytes. Spectrin, the major skeleton component, extracted at 0 degrees C from normal erythrocytes, was present primarily as tetramers and high molecular weight complexes. In contrast, spectrin extracted from HPP erythrocytes under identical conditions contained a significant amount of dimers with a concomitant decrease of tetramers. Furthermore, spectrin dimers from HPP erythrocytes differed from normal spectrin dimers in their failure to reassociate into tetramers both in solution and in the membrane. Presumptive HPP carriers (asymptomatic mothers of the two patients) exhibited a mild but reproducible increase of spectrin dimers in 0 degrees C extracts and a defective reassociation of spectrin dimers of tetramers both in solution and in the membrane. We conclude that in HPP, self-association of spectrin dimers into tetramers is defective, which accounts for the instability of membrane skeletons.

Evidence for an age cutoff greater than 365 days for neuroblastoma risk group stratification in the Children's Oncology Group.

PURPOSE: In the Children's Oncology Group, risk group assignment for neuroblastoma is critical for therapeutic decisions, and patients are stratified by International Neuroblastoma Staging System stage, MYCN status, ploidy, Shimada histopathology, and diagnosis age. Age less than 365 days has been associated with favorable outcome, but recent studies suggest that older age cutoff may improve prognostic precision. METHODS: To identify the optimal age cutoff, we retrospectively analyzed data from the Pediatric Oncology Group biology study 9047 and Children's Cancer Group studies 321p1-p4, 3881, 3891, and B973 on 3,666 patients (1986 to 2001) with documented ages and follow-up data. Twenty-seven separate analyses, one for each different age cutoff (adjusting for MYCN and stage), tested age influence on outcome. The cutoff that maximized outcome difference between younger and older patients was selected. RESULTS: Thirty-seven percent of patients were younger than 365 days, and 64% were > or = 365 days old (4-year event-free survival [EFS] rate +/- SE: 83% +/- 1% [n = 1,339] and 45% +/- 1% [n = 2,327], respectively; P < .0001). Graphical analyses revealed the continuous nature of the prognostic contribution of age to outcome. The optimal 460-day cutoff we selected maximized the outcome difference between younger and older patients. Forty-three percent were younger than 460 days, and 57% were > or = 460 days old (4-year EFS rate +/- SE: 82% +/- 1% [n = 1,589] and 42% +/- 1% [n = 2,077], respectively; P < .0001). Using a 460-day cutoff (assuming stage 4, MYCN-amplified patients remain high-risk), 5% of patients (365 to 460 days: 4-year EFS 92% +/- 3%; n = 135) fell into a lower risk group. CONCLUSION: The prognostic contribution of age to outcome is continuous in nature. Within clinically relevant risk stratification, statistical support exists for an age cutoff of 460 days.

Genetic staging of unresectable or metastatic neuroblastoma in infants: a Pediatric Oncology Group study.

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/m2 per day orally or intravenously on days 1-7) and doxorubicin (35 mg/m2 intravenously on day 8). Forty-two infants with diploid tumors (DNA index = 1.0; worse prognosis in retrospective studies) received cisplatin (90 mg/m2 intravenously on day 1) and teniposide (100 mg/ m2 intravenously on day 3) after an initial course of cyclophosphamide plus 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 hyperdiploid group (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.

Significance of chromosome 1p loss of heterozygosity in neuroblastoma.

We analyzed 156 primary neuroblastoma tumor samples for loss of heterozygosity at the distal short arm of chromosome 1 (1p LOH). We also compared 1p LOH with known clinical and genetic prognostic variables as well as patient outcome. 1p LOH was detected in 30 of 156 tumors (19%) and was strongly associated with adverse clinical and biological features. 1p LOH was also strongly predictive of a poor outcome in univariate analyses (estimated 4-year survival, 32 +/- 10% SE versus 76 +/- 5% SE; P < 0.001). However, the prognostic value of 1p LOH was equivocal when stratified for amplification of the MYCN oncogene (P = 0.16). We conclude that 1p LOH is an important component of a pattern of genetic abnormalities in neuroblastoma associated with an aggressive clinical course.

The Pediatric Oncology Group experience with the international staging system criteria for neuroblastoma. Member Institutions of the Pediatric Oncology Group.

PURPOSE: An international consensus on the criteria for surgicopathologic staging (INSS) of patients with neuroblastoma has been published, but has not been validated. A retrospective study was conducted to assess if the INSS definitions identified prognostic subsets of patients with neuroblastoma. PATIENTS AND METHODS: The initial operative and pathology reports were reviewed from 675 patients on Pediatric Oncology Group (POG) #8104, a stage- and age-related treatment study that used the POG surgicopathologic staging system. RESULTS: Of 596 eligible cases, there was concordance between the POG and INSS stages for the 193 patients with localized, resected disease (POG stage A), the 202 with distant metastases, the 51 with POG stage Ds (IVs) tumors, and 40 of the cases with grossly unresected, localized tumor without lymph node involvement (POG stage B). Of the remaining 19 patients with POG stage B tumors, five were INSS stage 2B and 14 INSS stage 3. All of the 91 cases with nonadherent, regional lymph node metastases (POG stage C) conformed to the definitions for INSS stage 2B (n = 42) or 3 (n = 49). In infants, there was no difference in event-free survival (EFS) among INSS stages 2A, 2B, or 3. In contrast, older children with INSS stage 3 disease had inferior EFS compared with INSS stage 2A or 2B tumors. CONCLUSION: We conclude the following: (1) the INSS identifies distinct patient subsets, particularly in children; (2) infants remain a favorable group, regardless of INSS/POG stage; and (3) the INSS deserves further prospective study especially in the light of recent biologic prognostic variables.

Circulating immune complexes in childhood malignancies: a Pediatric Oncology Group study.

Pretreatment serum samples obtained at diagnosis from 89 children with various pediatric malignancies were examined for circulating immune complexes (CIC) using the [125I]Clq binding assay. The study population consisted of 35 children with acute lymphocytic leukemia (ALL), 22 children with acute non-lymphocytic leukemia (ALL), 24 with neuroblastoma (NB), and eight with osteosarcoma (OS). Concomitant quantitation of immunoglobulins was performed in 55 patients, revealing normal values for age. Increased levels of CIC at diagnosis were found in 9%, 22%, 42%, and 50% of children with ALL, AML, NB, and OS, respectively. Except for a higher proportion of CIC-positive patients observed in stage IV NB (nine of 17) compared to stage I-III NB (one of seven), no correlation was observed between initial CIC level and presenting clinical features, response to treatment, prognosis, or presence of infection. Longitudinal sampling of six NB and two OS patients did not reveal a clear relationship between disease activity and quantity of CIC. For the pediatric malignancies studied, these data demonstrate minimal value in quantitating CIC as a means of assessing disease activity or predicting response to treatment and are in contrast to the apparently adverse effect of elevated pretreatment CIC on response to therapy and survival observed in adults with ALL, AML, and OS.

Clinical relevance of tumor cell ploidy and N-myc gene amplification in childhood neuroblastoma: a Pediatric Oncology Group study.

We assessed tumor cell DNA content (ploidy) and N-myc gene copy number as predictors of long-term disease-free survival in 298 children with neuroblastoma. Diploid tumor stem lines were identified in 101 patients (34%), clonal hyperdiploid abnormalities in 194 (65%), and hypodiploid stem lines in three (1%). In children with widely disseminated tumors at diagnosis (stage D), ploidy had a highly age-dependent influence on prognosis. Among infants (less than 12 months) treated with cyclophosphamide-doxorubicin, hyperdiploidy was closely associated with long-term disease-free survival (greater than 90% of cases), while diploidy invariably predicted early treatment failure (P less than .001). Similarly, in children 12 to 24 months of age who were treated with cisplatin-teniposide and cyclophosphamide-doxorubicin, diploidy uniformly predicted early failure, whereas half of the children with hyperdiploidy achieved long-term disease-free survival (P less than .001). There was no relationship between ploidy and treatment outcome in children older than 24 months with stage D tumors who had a very low probability of long-term disease-free survival (less than 10%). N-myc gene amplification was detected in 37 (25%) of the 147 tumors tested, with the remainder showing single-copy levels of the gene. N-myc gene amplification was more frequent in diploid than in hyperdiploid tumors (23 of 57 v 14 of 87, P = .001) and predicted a high likelihood of early treatment failure. In children younger than 2 years with disseminated neuroblastoma, tumor cell ploidy and N-myc gene copy number provide complementary prognostic information that will distinguish patients who can be cured on current regimens from those who require new treatment strategies.

International criteria for diagnosis, staging, and response to treatment in patients with neuroblastoma.

Neuroblastoma is one of the most common tumors in childhood. However, it often has been difficult to compare clinical and laboratory studies of this disease due to a lack of uniform criteria for diagnosis, staging, and response. An international group of conferees addressed each of these issues and reached a consensus. Specific criteria for making a diagnosis of neuroblastoma are defined. A new neuroblastoma staging system is proposed that takes into account the most important elements of current but incompatible systems. Finally, criteria for response to treatment are standardized. The criteria proposed herein represent an international consensus of essentially every major pediatric oncology group or organization in the United States, Europe, and Japan. The staging system should be referred to as the International Neuroblastoma Staging System, and the response criteria as the International Neuroblastoma Response Criteria. Implementation of these criteria will greatly facilitate the comparison of clinical and laboratory studies by different groups and countries. Furthermore, these criteria should serve as a foundation on which future modifications or improvements can be based.

Infants with neuroblastoma and regional lymph node metastases have a favorable outlook after limited postoperative chemotherapy: a Pediatric Oncology Group study.

PURPOSE: Infants less than or equal to 1 year of age with neuroblastoma (NB) have a favorable outlook with minimal to moderate therapy. Patients with complete or partial removal of the primary tumor but positive intracavitary lymph nodes (Pediatric Oncology Group [POG] stage C) have a higher risk for recurrent disease. To determine the importance of distinguishing infants with POG stage C NB from those with POG stage B disease and to assess the efficacy and toxicity of treating POG stage C infants with limited, postoperative chemotherapy, a study was conducted by the POG. PATIENTS AND METHODS: Forty-four eligible POG stage C infants received cyclophosphamide 150 mg/m2 orally on days 1 to 7 and Adriamycin (doxorubicin; Adria Laboratories, Columbus, OH) 35 mg/m2 intravenously (IV) on day 8 (CYC/ADR), every 3 weeks for five courses followed by second-look surgery. No continuation therapy was given if surgical and pathologic complete response (CR) was achieved. Secondary therapy with five courses of cisplatin 90 mg/m2 on day 1 followed by teniposide (VM-26) 100 mg/m2 on day 3 (CDP/VM) was given to infants with gross residual tumor after CYC/ADR and second-look surgery. RESULTS: Thirty-four infants achieved CR after CYC/ADR alone, three after CYC/ADR and second-look surgery, two after CYC/ADR, surgery, and maintenance therapy, and two after alternative treatment with CDP/VM (total CR rate, 42 of 44). The 3-year survival and disease-free survival are both 93%. Toxicity was nominal. CONCLUSIONS: Infants with POG stage C NB have a favorable outlook, which is similar to infants with POG stage B NB; the surgical staging procedure for distinguishing these infant subsets may not be necessary. Future studies should focus on the reduction of treatment toxicity and efficacy maintenance, and address methods to identify infants at risk for failure.

Radiotherapy improves the outlook for patients older than 1 year with Pediatric Oncology Group stage C neuroblastoma.

Children older than 1 year of age who have neuroblastoma with complete or partial removal of the primary tumor and positive intracavitary lymph nodes (Pediatric Oncology Group [POG] stage C) are a small but higher-risk subset of patients. To further evaluate the importance of identifying patients with POG stage C neuroblastoma and to assess the efficacy and toxicity of adding concurrent radiation therapy (RT) to chemotherapy (CT) in these children, a randomized study was conducted. Eligible patients received cyclophosphamide 150 mg/m2 orally days 1 to 7 and Adriamycin (doxorubicin; Adria Laboratories, Columbus, OH) 35 mg/m2 intravenously (IV) on day 8 (CYC/ADR) every 3 weeks for five courses with or without RT to primary tumor and regional lymph nodes (24 to 30 Gy/16 to 20 fractions). Second-look surgery was advised to evaluate response and to remove residual disease. Continuation therapy alternated CYC/ADR every 3 weeks with cisplatin 90 mg/m2 day 1 followed by teniposide 100 mg/m2 day 3 (CDP/VM) for two courses each. Secondary CT with CDP/VM alone was available for patients not achieving complete response (CR) following induction treatment and second-look surgery. Of 29 eligible patients randomized to CT alone, 13 achieved CR, and nine are disease-free (NED) 1 to 52 months (median, 35 months) off therapy. Twenty-two of 33 eligible cases treated with CT/RT attained CR, and 19 are NED 1 to 77 months (median, 23 months) off therapy. Local and metastatic relapses occurred in both arms. Differences in CR, event-free survival, and survival rates were significant, P = .013, .009, and .008, respectively. Surgical compliance was excellent and complications uncommon. Therapy was tolerable in both groups but hematopoietic toxicity was more common in the CT/RT arm. We conclude that POG stage C neuroblastoma in children older than 1 year of age is a higher-risk group that should be identified, that CT/RT provides superior initial and long-term disease control compared with CT alone in this patient subset, and that the occurrence of metastatic failures in both treatment groups suggests a need for more aggressive chemotherapy.

The prognostic significance of autologous bone marrow transplant in advanced neuroblastoma.

This report provides strong evidence for conducting a controlled randomized clinical trial of autologous bone marrow transplantation versus conventional chemotherapy in childhood neuroblastoma, which is disseminated beyond the intracavity nodes, and which is diagnosed in children older than 12 months of age. On the basis of two Pediatric Oncology Group (POG) studies, one a surgery plus conventional chemotherapy study (POG 8441) and the other an elective autologous transplant pilot protocol (POG 8340), there was no significant prognostic benefit of switching in remission from the surgery plus chemotherapy protocol to the transplant protocol (P = .91) or of switching in remission from the surgery plus chemotherapy protocol to any transplant (P = .75). The analysis is based on 116 patients achieving a complete or partial remission, 32 of whom received transplants on the pilot protocol, and 17 of whom received transplants outside the pilot protocol. While potential selection bias precludes cause-effect conclusions, these data strongly suggest that a large randomized trial of autologous bone marrow transplantation should be conducted before accepting this form of therapy as standard.

Phase II investigational window using carboplatin, iproplatin, ifosfamide, and epirubicin in children with untreated disseminated neuroblastoma: a Pediatric Oncology Group study.

PURPOSE: Children less than 1 year of age with metastatic neuroblastoma NB are at high risk of death. The need to identify new and effective chemotherapy agents is clear. A study was conducted by the Pediatric Oncology Group (POG) to determine the efficacy and safety of administering two courses of a single phase II agent before conventional treatment as a means to evaluate new agents in this setting. PATIENTS AND METHODS: One hundred seventy-three eligible patients more than 1 year of age with disseminated neuroblastoma received two courses of one of the following: ifosfamide (IFOS) 2 g/m2/d for 4 days intravenously (IV) plus mesna; carboplatin (CARB) 560 mg/m2 i.v. over 1 hour; iproplatin (CHIP) 325 mg/m2 IV over 2 hours; or epirubicin (EPIR) 90 mg/m2 i.v. push. Following evaluation for response and toxicity, eligible patients were randomized to receive either cisplatin 90 mg/m2 i.v. on day 1, etoposide 200 mg/m2 i.v. on day 3, cyclophosphamide 150 mg/m2/d orally on days 7 to 13, doxorubicin 35 mg/m2 i.v. on day 14 (CECA), or cisplatin 40 mg/m2 IV on days 1 to 5 and etoposide 200 mg/m2 i.v. on days 2 to 4 alternating at 3-week intervals with cyclophosphamide 150 mg/m2/d orally on days 1 to 7 and doxorubicin 35 mg/m2 IV on day 8 (HDP/VP/CA). An additional 86 patients were randomized to receive either CECA or HDP/VP/CA without initial phase II therapy. RESULTS: After phase II therapy, only 20% of patients experienced grade 3/4 hematopoietic toxicity. No toxic deaths occurred. Objective response rates (partial responses [PRs] plus minor responses [MRs]) following IFOS, CARB, CHIP, and EPIR were 70%, 77%, 67%, and 26%, respectively. Following phase III treatment, there was no statistically significant difference in rates of complete response (CR)/PR or progressive disease (PD), or in time to PD of patients who participated in the phase II window versus those who received only CECA or HDP/VP/CA. CONCLUSION: IFOS, CARB, and CHIP are efficacious in neuroblastoma, are well tolerated, and should be incorporated into primary treatment regimens. Combination regimens using these agents may be possible, since most repeat courses were given within 2 weeks. Administering phase II therapy to untreated patients with high-risk tumors provides a unique and sensitive method to assess new agents without compromising patient outcome.

Complete surgical excision is effective treatment for children with immature teratomas with or without malignant elements: A Pediatric Oncology Group/Children's Cancer Group Intergroup Study.

PURPOSE: To determine whether the 3-year event-free survival (EFS) of children with completely resected immature teratomas is greater than 85%. PATIENTS AND METHODS: Patients with immature teratomas treated at Pediatric Oncology Group or Children's Cancer Group institutions were eligible. Pathology was centrally reviewed to confirm diagnosis and tumor grading. Follow-up included physical examination, measurement of tumor markers (alpha fetoprotein and human chorionic gonadotropin), and imaging. All patients were monitored for events, defined as tumor recurrence, second malignancy, or death. RESULTS: Seventy-three children (median age, 7.8 years) with extracranial immature teratomas were enrolled on study. Primary tumor sites included ovarian (n = 44), testicular (n = 7), and extragonadal (n = 22). However, on review, 23 patients had foci of yolk sac tumor (n = 21) or primitive neuroectodermal tumor (n = 2), whereas 50 had pure immature teratomas. Twenty-five patients had increased alpha fetoprotein (n = 18), human chorionic gonadotropin (n = 5), or both (n = 2); nine had foci of yolk sac tumor on review. Pathology review identified 23 patients with grade 1, 29 with grade 2, and 21 with grade 3 immature teratomas. With a median follow-up of 35 months, the overall 3-year EFS was 93% (95% confidence interval, 86% to 98%), with 3-year EFS of 97.8%, 100%, and 80% for patients with ovarian, testicular, and extragonadal tumors, respectively. Only four of 23 patients with immature teratoma and malignant foci developed recurrence, suggesting that surgical resection followed by close observation are effective treatment. Overall, five patients had disease recurrence 4 to 7 months from diagnosis, and four (80%) are disease free after platinum-based therapy. The fifth patient has residual tumor after cisplatin, etoposide, and bleomycin treatment requiring further therapy. CONCLUSION: Surgical excision is safe and effective treatment for 80% to 100% of children with immature teratoma.

Prognostic significance of age, MYCN oncogene amplification, tumor cell ploidy, and histology in 110 infants with stage D(S) neuroblastoma: the pediatric oncology group experience--a pediatric oncology group study.

PURPOSE: Although a high rate of spontaneous regression is observed in infants with stage D(S) neuroblastoma (NB), survival is not uniform. To determine the prognostic relevance of age at diagnosis, therapy, and tumor biology in infants with stage D(S) NB, we reviewed the Pediatric Oncology Group (POG) experience. PATIENTS AND METHODS: A review of patients diagnosed with stage D(S) NB registered on POG protocols was performed. Survival according to age at diagnosis, treatment, and tumor biology was determined. RESULTS: Between 1987 and 1996, 110 infants with stage D(S) NB had an estimated 3-year survival rate of 85% +/- 4%; survival rate was 71% +/- 8% for infants 2 months of age or younger, and 68% +/- 12%, 44% +/- 33%, and 33% +/- 19% for patients with diploid, MYCN-amplified, and unfavorable histology tumors, respectively. Survival rates were similar for patients who received adjuvant chemotherapy versus those who did not (82% +/- 5% v 93% +/- 6%, respectively; P = .187). Furthermore, there was no statistical difference in survival rate for patients who underwent complete resection of their primary tumor compared with those who underwent partial resection or biopsy only (90% +/- 5% v 78% +/- 7%, respectively; P = .083). CONCLUSION: Our review confirmed that the survival of infants with stage D(S) NB is excellent. However, subsets of patients with poor prognosis can be identified by young age and unfavorable biologic factors. More effective therapy is needed for the group of stage D(S) infants who show unfavorable clinical and biologic features.

Cell culture studies and oncogene expression in juvenile chronic myelogenous leukemia.

Juvenile chronic myelogenous leukemia (JCML) may be distinguished from adult CML based upon in vitro cell growth characteristics. We studied four untreated children with JCML and report additional unique findings. Peripheral blood (PB) and bone marrow (BM) cells were grown in soft agar. Without exogenous colony-stimulating activity (CSA) there was exuberant "spontaneous" colony formation in both PB and BM cultures. In the absence of exogenous stimulus, PB colony morphology was predominantly, but not exclusively, monocyte/macrophage. When PB was depleted of adherent cells, "spontaneous" colony formation was nearly completely abrogated. Cultures were also performed in the presence of various sources of CSA including giant cell tumor-conditioned medium (GCT-CM), a melanoma cell line-CM (LD1-CM), human placenta-CM (HPCM), and normal PB mononuclear cell (PBMC) feeder layers. Colony formation was typically increased with HPCM and PBMC, whereas in two patients GCT-CM and LD1-CM failed to stimulate additional colony growth when compared to cultures without exogenous CSA and, in fact, appeared to inhibit baseline "spontaneous" growth. The morphology of colonies in the presence of exogenous stimuli was highly variable. Because of the recent association between the c-fms protooncogene product and the receptor for the monocyte growth factor CSF-1, we analyzed the PB cells from two JCML patients for c-fms expression. Although expressed, c-fms levels were less than that in an adult with Ph1-positive CML in chronic phase. These studies indicate that in JCML, there are dramatic increases in both PB and BM colony-forming cells and that "spontaneous" growth is dependent on an accessory adherent cell fraction. Furthermore, patterns of responsiveness to various sources of CSA suggest that the colony-forming cells may not be a uniform population of malignant cells.

The role of monocyte-derived hemopoietic growth factors in the regulation of myeloproliferation in juvenile chronic myelogenous leukemia.

Juvenile chronic myelogenous leukemia (JCML) is a rare pediatric malignancy characterized by marked hepatosplenomegaly, leukocytosis with prominent monocytosis, elevated fetal hemoglobin, no Philadelphia chromosome, and generally a poor prognosis. In vitro, JCML peripheral blood granulocyte-macrophage progenitors (granulocyte-macrophage colony-forming units, CFU-GM) demonstrate the unique characteristic of "spontaneous" proliferation at very low cell densities in the absence of exogenous growth factors. The "spontaneous" CFU-GM proliferation can be abolished by prior adherent cell (monocyte) depletion, suggesting a paracrine mode of cellular proliferation. Although previous studies using a [3H]thymidine ([3H]TdR) incorporation assay suggested an important role for granulocyte-macrophage colony-stimulating factor (GM-CSF) in JCML, many non-growth factor-related reasons for [3H]TdR incorporation and the relatively low level of inhibition of [3H]TdR uptake left those conclusions open to question. Therefore, we performed clonal CFU-GM assays, which more specifically reflect cytokine effects on CFU-GM, using JCML peripheral blood mononuclear cells (PBMNC) and neutralizing antibodies against GM-CSF, granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating (M-CSF), interleukin 3 (IL-3), interleukin 1 alpha (IL-1 alpha), interleukin 1 beta (IL-1 beta), interleukin 4 (IL-4), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF alpha), and interferon gamma (IFN gamma). Cultures containing anti-GM-CSF alone inhibited "spontaneous" JCML CFU-GM by 87% +/- 9% (mean +/- standard error of the mean [SEM]). No other anti-cytokine antibody produced a significant inhibition of CFU-GM growth. Various combinations of antibodies, excluding anti-GM-CSF, failed to demonstrate any synergistic inhibitory effects upon CFU-GM. Because this apparent paracrine cellular stimulation could be due to excessive cytokine production, by monocytes or other accessory cells, we examined cytokine levels in conditioned media from various JCML cell populations using enzyme-linked immunosorbent assays (ELISAs). Monocytes from only a minority of JCML patients produced higher than normal quantities of GM-CSF, G-CSF, IL-1 beta, IL-6, and/or TNF alpha, but no obvious pattern could be discerned. Further, only 7 of 15 JCML monocyte-conditioned media (MCM) had elevated GM-CSF, and 6 of 15 JCML patients had normal levels of all nine cytokines tested. The monocyte depletion experiments and the inhibition experiments with anti-cytokine antibodies taken together demonstrate clearly that the "spontaneous" growth of JCML CFU-GM in vitro critically depends on at least one monocyte-derived growth factor, GM-CSF.(ABSTRACT TRUNCATED AT 400 WORDS)

Detection of MYCN gene amplification in neuroblastoma by fluorescence in situ hybridization: a pediatric oncology group study.

To assess the utility of fluorescence in situ hybridization (FISH) for analysis of MYCN gene amplification in neuroblastoma, we compared this assay with Southern blot analysis using tumor specimens collected from 232 patients with presenting characteristics typical of this disease. The FISH technique identified MYCN amplification in 47 cases, compared with 39 by Southern blotting, thus increasing the total number of positive cases by 21%. The major cause of discordancy was a low fraction of tumor cells (< or =30% replacement) in clinical specimens, which prevented an accurate estimate of MYCN copy number by Southern blotting. With FISH, by contrast, it was possible to analyze multiple interphase nuclei of tumor cells, regardless of the proportion of normal peripheral blood, bone marrow, or stromal cells in clinical samples. Thus, FISH could be performed accurately with very small numbers of tumor cells from touch preparations of needle biopsies. Moreover, this procedure allowed us to discern the heterogeneous pattern of MYCN amplification that is characteristic of neuroblastoma. We conclude that FISH improves the detection of MYCN gene amplification in childhood neuroblastomas in a clinical setting, thus facilitating therapeutic decisions based on the presence or absence of this prognostically important biologic marker.

Neuroblastoma.

The neuroblastic tumours, derived from primordial neural crest cells which ultimately populate the sympathetic ganglia, adrenal medulla and other sites, (Brodeur GM and Castleberry RP. Neuroblastoma. In Pizzo PA, Poplack DG, eds, Principles and Practice of Pediatric Oncology. Philadelphia, J. B. Lippincott Co., 1997, 761-797) are an enigmatic group of neoplasms which have the highest rate of spontaneous regression of all human malignant neoplasms yet one of the poorest outcomes when occurring as disseminated disease in children. Significant advances in understanding and predicting the natural history of neuroblastoma have resulted from translational studies coupling tumour biology and clinical features to form prognostic strata and allowing more accurate routeing of patients to risk-related management. While this strategy has clarified the management for lower risk tumours, little improvement in survival for higher risk disease has been realised. Ironically, this latter patient subset, for which the most innovative therapeutic strategies are needed, is also the one from which the least tumour biology is gleaned owing to inadequate tissue sampling. This update will summarise the evolving biology of neuroblastoma and its relationship to current risk-related therapy and future management strategies. Throughout this report, prognostic grouping by age will be infants (< 1 year) versus children (> or = 1 year) since the change of risk according to age seems most distinct at this cut-off point.

Event-free survival of children with biologically favourable neuroblastoma based on the degree of initial tumour resection: results from the Pediatric Oncology Group.

We analysed the 2-year event-free survival (EFS) of 49 patients 1 year of age and older, with stage 2B or 3 neuroblastoma, treated on Pediatric Oncology Group protocols 8742 and 9244, with respect to the degree of tumour resection at diagnosis. The 2-year EFS rate for 21 children whose tumours were completely resected at diagnosis was 85% (SE = 10%) compared with an EFS rate of 70% (SE = 9%) for the 28 children whose tumours were incompletely resected at diagnosis. Despite the observed trend in favour of complete resection, these EFS curves were not statistically significantly different (P = 0.259). Patients with favourable Shimada histology tumours had an EFS rate of 92% (SE = 7%) compared with a rate of 58% (SE = 15%) for patients with unfavourable histology tumours. EFS curves for the two histologic groups were significantly different (P = 0.009). The impact of aggressive surgery and adjuvant chemotherapy on the outcome of patients with biologically favourable regional neuroblastoma is still unclear.

Accidental poisoning with a superwarfarin compound (brodifacoum) in a child.

The "superwarfarin" compounds are 4-hydroxy derivatives of coumarin that have increased activity and a longer duration of action than the parent compound. The superwarfarins are used widely in the United States as rodenticides and are effective against warfarin-resistant strains of rats. A chronic accidental ingestion of one of these products, brodifacoum, by a 7-year-old child who had bleeding and laboratory evidence suggestive of a vitamin K-related coagulopathy is reported. The bleeding manifestations were severe and prolonged, requiring 13 months for normalization of coagulation times. With a negative history of ingestion and despite clinical suspicion, documentation of superwarfarin poisoning was hampered by the lack of readily available assays for these compounds, even from the manufacturers. Brodifacoum was also identified in rat feces from the family home. This finding raises the concern of poisoning not only from ingestion of brodifacoum particles themselves, but also from a fecal-oral route. A review of the literature is presented and the implications of this case for the practicing physician are discussed.