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Background The N7 protocol for poor-risk neuroblastoma uses dose-intensive chemotherapy (as in N6 protocol [Kushner et al.: J Clin Oncol 12:2607–2613, 1994] but with lower dosing of vincristine) for induction, surgical resection and 2100 cGy hyperfractionated radiotherapy for local control, and for consolidation, targeted radioimmunotherapy with 131I-labeled anti-GD2 3F8 monoclonal antibody and immunotherapy with unlabeled/unmodified 3F8 (400 mg/m2). Procedure The chemotherapy consists of: cyclophosphamide 70 mg/kg/d × 2 and a 72-hr infusion of doxorubicin 75 mg/m2 plus vincristine 2 mg/m2, for courses 1, 2, 4, and 6; and cisplatin 50 mg/m2/d × 4 and etoposide 200 mg/m2/d × 3, for courses 3, 5, and 7. 131I-3F8 is dosed at 20 mCi/kg, which is myeloablative and therefore necessitates stem-cell support. Results Of the first 24 consecutive previously untreated patients more than 1 year old at diagnosis, 22 were stage 4 and two were unresectable stage 3 with MYCN amplification. Chemotherapy achieved CR/VGPR in 21 of 24 patients. Twenty patients to date have completed treatment with 131I-3F8, and 15 patients have completed all treatment. With a median follow-up of 19 months, 18 of 24 patients remain progression-free. Conclusions Major toxicities were grade 4 myelosuppression and mucositis during chemotherapy, and self-limited pain and urticaria during antibody treatment. Late effects include hearing deficits and hypothyroidism. Med. Pediatr. Oncol. 36:227–230, 2001. © 2001 Wiley-Liss, Inc.

Background The recommended dosages of topotecan and cyclophosphamide in combination for prior-treated patients—3.75 mg/m2 and 1,250 mg/m2 in children, 5 mg/m2 and 600 mg/m2 in adults, respectively—are well below those of each agent when used singly. We tested the hypothesis that much higher dosing would meet critical goals of salvage therapy: antitumor effect and a lack of toxicity to key organs, so as not to preclude subsequent consolidative treatments needed for cure. Procedure Patients with resistant pediatric solid tumors received cyclophosphamide 4,200 mg/m2 by 48 hr infusion, and topotecan 6 mg/m2 by 72 hr infusion (HD-Cy/Topo). Mesna and granulocyte colony-stimulating factor were used. Cycles were repeated when neutrophil counts were >1,000/uL and platelet counts were >75,000/uL. Results Twenty-eight patients, aged 2 to 33 years (median, 14), received one (n = 4), two (n = 15), or ≥3 (n = 9) cycles of HD-Cy/Topo. All patients had previously received ≥6 cycles of other therapy, high-dose alkylator-based chemotherapy, and/or etoposide- and doxorubicin-containing regimens. HD-Cy/Topo was given in an outpatient setting. Profound myelosuppression was the major toxicity, but retreatment was possible by day 28, and preliminary results with peripheral blood stem cell collections showed a sparing effect on hemopoietic stem cells. Mucositis was uncommon. After HD-Cy/Topo, cardiac, renal, hepatic, and pulmonary function remained within the normal range. Partial or minor responses were noted in neuroblastoma, desmoplastic small round-cell tumor, Ewing sarcoma, rhabdomyosarcoma, and osteosarcoma. Conclusions Its antitumor potential and limited toxicity make HD-Cy/Topo an attractive choice for inclusion in aggressive salvage programs aimed at achieving cures of resistant tumors. It may also merit incorporation into frontline treatment protocols. Med. Pediatr. Oncol. 35:468–474, 2000. © 2000 Wiley-Liss, Inc.

Background Standard cytogenetic techniques are time-consuming and often not informative with solid tumors. In contrast, the reverse transcriptase-polymerase chain reaction (RT-PCR) is a readily available technique that can rapidly detect tumor-specific chromosomal rearrangements, even in small biopsy specimens. We present cases depicting the importance of including molecular diagnostic studies in the routine evaluation of pediatric solid tumors. Procedure We used RT-PCR to detect chimeric transcripts specific for major pediatric solid tumors, including peripheral primitive neuroectodermal tumor (pPNET), alveolar rhabdomyosarcoma (ARMS), and desmoplastic small round-cell tumor (DSRCT). We reviewed six recent cases in which the initial diagnosis was changed by the results of RT-PCR. Results Highly unusual or nonspecific clinical and/or histopathologic findings led to the initial diagnoses of neuroblastoma in three patients and DSRCT, leukemia, and carcinoma in one patient each. The final diagnoses after RT-PCR studies were pPNET in three patients, ARMS in two patients, and DSRCT in one patient. RT-PCR results led to early corrections in the diagnosis in two patients, but four patients received treatment not considered optimal for the neoplasms ultimately diagnosed, including three who, despite presenting with localized tumors that have a >70% cure rate with standard therapy, have died or are dying of disease. Conclusions Molecular genetic studies on solid tumors can clarify the diagnosis in seemingly straightforward as well as in overtly problematic cases. These diagnostic distinctions are now critical as disease-specific and risk-directed therapies have emerged. Med. Pediatr. Oncol. 33:530–535, 1999. © 1999 Wiley-Liss, Inc.

Background The identification of recently described nonrandom chromosomal defects specific for various small round-cell and spindle-cell sarcomas can eliminate diagnostic uncertainty arising from the clinical and histopathologic overlap of soft tissue neoplasms. Methods A 26-year-old man presented with bulky abdominal-pelvic disease. Immunohistochemical and molecular studies on tumor were performed. Treatment was instituted using cycles of high-dose cyclophosphamide (4,200 mg/m2) with doxorubicin (75 mg/m2). Results Clinical findings pointed to desmoplastic small round-cell tumor. The tumor was histologically undifferentiated and immunoreactive for vimentin but negative for other markers. Reverse transcriptase-polymerase chain reaction revealed the SYT/SSX2 fusion transcript of the synovial sarcoma t(X;18) chromosomal rearrangement. The high-dose chemotherapy, plus surgery, achieved a complete remission, but recurrent disease emerged 13 months from diagnosis. Conclusions This clinically unique case of synovial sarcoma highlights how the use of now readily available molecular techniques will allow more accurate appraisals of the incidence and anatomic distribution of soft tissue neoplasms—information that bears upon pathogenesis and treatment. This case confirms the utility of high-dose alkylator-based therapy for synovial sarcoma. It also demonstrates that with nonlocalized solid tumors, the eradication of minimal residual disease remains an elusive goal. One alternative involves immunologic attack against markers derived from tumor-specific chromosomal defects such as those found in our patient. Med. Pediatr. Oncol. 32:97–101, 1999. © 1999 Wiley-Liss, Inc.

Background Except at diagnosis and relapse, when gross disease is present, histologic evaluation is less sensitive than immunocytology (IC) of bone marrow for detecting metastatic neuroblastoma. We examined whether the chance of a positive IC from a single marrow site was comparable to an IC of pooled marrow from multiple sites. Procedure We carried out 47 marrow examinations on 29 patients with high-risk neuroblastoma on therapy. Each examination consisted of histologic evaluation of four aspirates and two biopsies (six sites), IC of a 2.5–5-mL heparinized sample from a single site (the right posterior iliac crest; IC-RPIC), as well as IC of 8–10 mL of heparinized marrow pooled from bilateral anterior and bilateral posterior iliac crests (IC-pooled). IC was performed using a panel of monoclonal antibodies specific for ganglioside GD2. Results The number of GD2-positive tumor cells detected by IC-pooled had a strong linear correlation with that by IC-RPIC (R = 0.91), although IC-pooled detected an average of 3.3 times more GD2-positive cells. Of 47 examinations, 15 tested positive by histology (6 sites), 20 by IC- pooled, and 17 by IC-RPIC. Among 29 patients, the level of agreement between IC-RPIC and IC-pooled was generally good (kappa statistic ⩾ 0.72), giving a false negative rate of ⩽30%. Conclusions Compared to conventional histologic examinations, immunocytology of a single marrow aspirate generally agrees with that of marrow pooled from six sites. However, the false negative rate may be too high in the setting of examination prior to bone marrow or stem cell harvest. Med. Pediatr. Oncol. 32:84–87, 1999. © 1999 Wiley-Liss, Inc.

Background Gangliosicle GD2 is abundant on human neuroblastoma (NB). Monoclonal antibody 3F8 targeted to GD2 may have imaging and therapeutic potential. Antigen-negative clones can escape immune-mediated attack leading to clinical resistance or recurrence. Procedure Among 95 evaluable patients treated intravenously with 3F8 (94 Stage 4, 1 Stage 3), 66 received nonradiolabeled 3F8, 11 received 131-iodine-labeled-3F8 (8-28 mCi/kg) with autologous bone marrow rescue, and 18 received both forms of treatment. Prior to treatment, 90 patients tested positive for GD2 reactivity by bone marrow immunofluorescence (n = 68), tumor immunohistochemistry (n = 20), or diagnostic radioimmunoscintigraphy (n = 2). Results Of 62 patients who had refractory or recurrent neuroblastoma following 3F8 treatment, 61 (98%) tested positive for GD2 reactivity by bone marrow immunofluorescence (n = 51) or tumor immunohistochemistry (n = 10). The sole tumor that lost GD2 expression underwent phenotypic transformation into a pheochromocytoma-like tumor. Conclusions The persistence of GD2 expression in refractory or recurrent NB suggests that complete antigen loss is an uncommon event and cannot account for treatment failure.

Background We first explored the use of multiple molecular markers to overcome tumor heterogeneity. Sixty-seven neuroblastoma (NB) tumors were tested for the expression of GAGE, MAGE-1, MAGE-2, MAGE-3, and MAGE-4 by RT-PCR and then chemiluminescence; 82% of tumors had detectable GAGE, and 88% expressed at least one of the four MAGE genes. Procedure and Results By combining GAGE and MAGE, 64 of 67 (95%) of tumors became detectable; 17 of 67 coexpressed all five molecular markers. Neither GAGE nor MAGE expression correlated with stage. GAGE was found to have the broadest (18 of 18) expression among stage 4 tumors. Two hundred fifty-nine bone marrows from 99 patients were then studied for NB positivity by four detection methods: histology, immunocytology, and molecular detection by GAGE and tyrosine hydroxylase (TH) mRNA. Two hundred seven samples were NB-positive by one detection method. All four techniques were comparable in detecting tumor cells at diagnosis and at relapse. GAGE and immunocytology were far more sensitive than histology and TH mRNA when marrows were sampled during chemotherapy and at the time of clinical remission. Conclusions By combining multiple molecular markers and independent screening techniques, we may be able to overcome tumor heterogeneity and expedite the detection of microscopic disease in the clinical management of neuroblastoma. Med. Pediatr. Oncol. 36:210–212, 2001. © 2001 Wiley-Liss, Inc.

Background EPH family receptor tyrosine kinases and their ligand ephrins play pivotal roles in development. High-level expression of transcripts encoding EPHB6 receptors (EPHB6), its ligands ephrin-B2 and ephrin-B3 (EFNB2, EFNB3) is predictive of favorable disease outcome of neuroblastoma (NB). When combined with TrkA expression, the expression of EPHB6, EFNB2, or EFNB3 predicts more accurately the disease outcome than each of the four variables alone. Procedure Cox regression and Kaplan-Meier analyses were used to assess the prognostic significance of EPHB6, EFNB2, EFNB3, and TrkA expressions in NB without MYCN amplification. Results High-level expression of EFNB3 or TrkA predicted favorable NB outcome of NB without MYCN amplification (p < 0.03). As found in the general NB population, EPHB6, EFNB2, or EFNB3 expression in combination with TrkA expression was significantly predictive of the disease outcome of normal MYCN NB (p < 0.01). Conclusions EPHB6, EFNB2, and EFNB3 expressions may permit further refinement of the prognostic stratification of NB into favorable and unfavorable groups. Med. Pediatr. Oncol. 35:656–658, 2000. © 2000 Wiley-Liss, Inc.

Background A transient human anti-mouse antibody response was associated with significantly longer survival [Cheung et al. (1998): J Clin Oncol 16:3053] following antibody 3F8 (Ab1) treatment. We postulate that the induction of an idiotype network which included anti-anti-idiotypic (Ab3) and anti-G(D2) (Ab3') responses is associated with tumor control. Procedure Thirty-four patients with stage 4 neuroblastoma (NB) diagnosed at > 1 year of age were treated with anti-G(D2) monoclonal antibody 3F8 at the end of chemotherapy Results Long-term progression-free survival and overall survival correlated significantly with Ab3' andAb3, but not with non-idiotypic antibody responses. Only one of six individual specificities showed significant correlations with patient survival. Conclusions As in vitro correlates of idiotype network initiated by Ab1 treatment, Ab3 and Ab3' may provide convenient biologic endpoints for monoclonal antibody therapy of advanced NB, and a rationale for choosing specific anti-idiotypic antibodies for vaccine development.

Background Intrathecal antibody-based targeted therapies may have clinical potential for patients with leptomeningeal (LM) cancer. Procedure Five patients with GD2-positive LM tumors were injected with 1-2 mCi intra-Ommaya (131)I-3F8, a murine IgG3 antibody specific for GD2. Serial cerebrospinal fluid (CSF) and serum samples and SPECT imagings (4, 24, and 48 hr) were performed to predict radiation doses to the tumor and normal brain and blood prior to the administration of larger therapeutic doses. Results Side effects included self-limited fever, headache, and vomiting. Focal (131)I-3F8 uptake consistent with tumors was seen along the craniospinal axis in four patients. Calculated radiation dose to the CSF was 14.9-56 cGy/mCi and to blood and other organs outside the CNS less than 2 cGy/mCi. Conclusions Intraventricular (131)I-3F8 successfully detected LM disease and resulted in a large favorable CSF/blood ratio. Intraventricular (131)I-3F8 may have clinical utility in the diagnosis and radioimmunotherapy of GD2-positive LM cancers. Med. Pediatr. Oncol. 35:716-718. 2000.

BACKGROUND Previously, we reported the utility of GAGE as a molecular marker for neuroblastoma (NB) and malignant melanoma in blood and bone marrow (BM). Among patients with stage III melanoma rendered disease-free by surgery, GAGE expression was a strong prognostic factor for patient survival. PROCEDURE All patients with advanced NB diagnosed at > 1 year of age initially treated with protocol N6 (n = 24) and N7 (n = 38) at Memorial Sloan-Kettering Cancer Center were included in this study. Their BM cells at 12, 18, and 24 months (median time after diagnosis) were evaluated for the presence of GAGE. RESULTS GAGE positivity at 12 months (25%), when patients were still on treatment, did not predict progression-free survival (PFS) and overall survival from the time of sampling. Positivity at 18 months (29%) was associated with poorer PFS and survival (but P > 0.05). By 24 months, the presence of GAGE (26%) was a very strong predictor of out-come (P < 0.001). When only remission marrows at 24 months were analyzed, PFS was 4.7-fold lower among GAGE-positive patients. Thirty-seven percent of N6 patients were positive for GAGE, in contrast to 17% of the patients in the more current regimen N7. CONCLUSIONS The detection of GAGE by RT-PCR in marrow may have utility in molecular staging of patients in clinical remission. It may allow earlier identification of patients at risk, such that appropriate intervention can be given before clinical relapse. GAGE may also serve as a surrogate endpoint for adjuvant treatment strategies, and to determine the duration of therapy.

Intensive chemotherapy prior to harvesting autologous bone marrow may be advantageous for cancers that infiltrate the bone marrow. This approach, however, may deplete hemopoietic reserves or damage the hemopoietic microenvironment and thereby jeopardize posttransplant engraftment; in vitro treatment of bone marrow to ensure a tumor-free state may enhance the risk of nonengraftment. We addressed these concerns in a pilot study of patients with disseminated neuroblastoma. Bone marrow was harvested after initial intensive therapy that included 1) three to six courses of very high dose cyclophosphamide (CPM) (100-140 mg/kg in five patients, 140-160 mg/kg in nine patients), plus doxorubicin and vincristine, followed by 2) a median of three strongly myelosuppressive courses of cisplatin/VP16. The median interval between courses was 23 days. The marrow was treated in vitro with the CPM congener 4-hydroperoxycyclophosphamide (4-HC) and cryopreserved. It was infused after a massive cytoreduction regimen of melphalan 180 mg/m2 (n = 7) or thiotepa 900 mg/m2 (n = 7), plus cisplatin, BCNU, VP16, and local radiation. All 14 patients reconstituted hemopoiesis (median of 37 days to neutrophils greater than or equal to 500/microliter and of 63 days to platelets greater than 50,000/microliters). The number per kilogram body weight of hemopoietic progenitor cells (CFU-GM, BFU-E) in the autografts correlated significantly with the rate of hematologic recovery. Preharvest CPM dosage, however, did not. The use of thiotepa, as opposed to melphalan, was associated with a significantly slower posttransplant platelet recovery, suggesting a possible adverse effect of high-dose thiotepa on posttransplant hemopoiesis. In sum, while reinforcing evidence of a stem cell sparing effect of CPM, this pilot study more importantly delineates the degree to which high-dose alkylator therapy can be exploited to attain maximal dose intensity beginning at diagnosis. This aggressive treatment approach may have wide applicability, since CPM is among the most active agents against a spectrum of pediatric cancers.

Objective To compare the long-term myocardial function of patients who had been treated with infusion anthracycline therapy (administered continuously over >24 hr, IG) versus bolus therapy (administered over

Background Desmoplastic small round cell tumor (DSRCT) is an aggressive and often misdiagnosed neoplasm of children and young adults. It is chemotherapy-sensitive, yet patients often relapse off therapy because of residual microscopic disease at distant sites: peritoneum, liver, lymph node, and lung. Strategies directed at minimal residual disease (MRD) may be necessary for cure. Monoclonal antibodies selective for cell surface tumor-associated antigens may have utility for diagnosis and therapy of MRD, as recently demonstrated in advanced-stage neuroblastoma (JCO 16: 3053, 1998). We examined DSRCT samples for the expression of two tumor antigens that could serve as possible targets for antibody-based immunotherapeutic approaches. Procedures Using immunohistochemistry, we studied the expression of two antigens: (1) GD2 using antibody 3F8 and (2) a novel antigen using antibody 8H9 in a panel of 46 freshly frozen DSRCT. GD2 is a disialoganglioside, which is widely expressed among neuroectodermal tumors as well as adult sarcomas. 8H9 recognizes a 58 kDa surface antigen expressed among neuroectodermal, mesenchymal, and epithelial tumors with restricted expression on normal tissues. Results Thirty-two of 46 (70%) tumors were reactive with 3F8, and 44 of 46 (96%) with 8H9. Both GD2 and the 58 kDa antigen were localized to tumor cell membrane and stroma. In general, immunoreactivity was stronger and more homogeneous with 8H9 than with 3F8. There was no correlation between expression of either antigen or clinical outcome. Conclusions GD2 and the novel tumor antigen recognized by 8H9 are potential targets for immunodiagnosis and antibody-based therapy of DSRCT. Med Pediatr Oncol 2002;39:547–551. © 2002 Wiley-Liss, Inc.

BACKGROUND Neuroblastoma (NB) is a heterogeneous tumor composed of mixed populations of neuroblastic, Schwannian, and gangliocytic cells. PROCEDURE We examined the genetic lineage of Schwannian and neuroblastic cells in stage 4 NB using laser-capture microdissection (LCM) and allelic analysis of microsatellite markers of chromosomes 1p, 11q, and 14q. RESULTS In 18 out of 19 cases, individual cell types exhibited the same allelotype. CONCLUSION Our results provide evidence that the majority of Schwannian and neuroblastic cells in stage 4 NB are of neoplastic origin, and support the hypothesis of a tumor stem cell capable of development along multiple lineages.