Claudia Sampor

Detection of N-Glycolyl GM3 Ganglioside in Neuroectodermal Tumors by Immunohistochemistry: An Attractive Vaccine Target for Aggressive Pediatric Cancer

The N-glycolylated ganglioside NeuGc-GM3 has been described in solid tumors such as breast carcinoma, nonsmall cell lung cancer, and melanoma, but is usually not detected in normal human cells. Our aim was to evaluate the presence of NeuGc-GM3 in pediatric neuroectodermal tumors by immunohistochemistry. Twenty-seven archival cases of neuroblastoma and Ewing sarcoma family of tumors (ESFT) were analyzed. Formalin-fixed, paraffin-embedded tumor samples were cut into 5 μm sections. The monoclonal antibody 14F7, a mouse IgG1 that specifically recognizes NeuGc-GM3, and a peroxidase-labeled polymer conjugated to secondary antibodies were used. Presence of NeuGc-GM3 was evident in 23 of 27 cases (85%), with an average of about 70% of positive tumors cells. Immunoreactivity was moderate to intense in most tumors, showing a diffuse cytoplasmic and membranous staining, although cases of ESFT demonstrated a fine granular cytoplasmic pattern. No significant differences were observed between neuroblastoma with and without NMYC oncogene amplification, suggesting that expression of NeuGc-GM3 is preserved in more aggressive cancers. Until now, the expression of N-glycolylated gangliosides in pediatric neuroectodermal tumors has not been investigated. The present study evidenced the expression of NeuGc-GM3 in a high proportion of neuroectodermal tumors, suggesting its potential utility as a specific target of immunotherapy.

Outcome of Children With Retinoblastoma and Isolated Choroidal Invasion

OBJECTIVES To evaluate the outcome of children with different degrees of choroidal invasion, to compare different systems for grading the extent of choroidal invasion, and to assess the role of concomitant prelaminar optic nerve and anterior segment invasion as predictors of extraocular relapse. DESIGN Retrospective analysis of children included in 4 prospective protocols (January 1, 1989, through June 31, 2010). Children with postlaminar optic nerve or scleral involvement and overt extraocular disease were excluded. Adjuvant chemotherapy was not scheduled. All slides were reviewed, and massive involvement was classified according to 3 definitions: (1) extending at least 3 mm in any dimension, (2) through the choroids whole thickness, and (3) more than 50% of the thickness and/or more than 1 cluster. RESULTS One hundred sixty-seven children (35 with massive invasion) were studied (136 did not receive adjuvant therapy). The probability of 5-year event-free survival was 98.1% and the probability of overall survival was 98.7% because 1 patient relapsed. Children with massive invasion had a significantly lower event-free survival probability (94.2%) compared with those with focal invasion (99.2%) (P = .04). However, no significant difference was found in overall survival probability (98.7% vs 99.2%; P = .29). No significant effect of other risk factors was found. CONCLUSIONS Survival was excellent without adjuvant therapy, and no other factors correlated with survival. Children with massive invasion have a higher relapse rate but comparable survival to those with focal invasion provided that aggressive therapy for extraocular relapse is available with adequate safety conditions.

Clinical Pharmacokinetics of Intra-arterial Melphalan and Topotecan Combination in Patients with Retinoblastoma

PURPOSE To assess the antitumor activity, toxicity, and plasma pharmacokinetics of the combination of melphalan and topotecan for superselective ophthalmic artery infusion (SSOAI) treatment of children with retinoblastoma. DESIGN Single-center, prospective, clinical pharmacokinetic study. PARTICIPANTS Twenty-six patients (27 eyes) with intraocular retinoblastoma. METHODS Patients with an indication for SSOAI received melphalan (3-6 mg) and topotecan (0.5-1 mg; doses calculated by age and weight). Plasma samples were obtained for pharmacokinetic studies, and a population approach via nonlinear mixed effects modeling was used. Safety and efficacy were assessed and compared with historical cohorts of patients treated with melphalan single-agent SSOAI. MAIN OUTCOME MEASURES Melphalan and topotecan pharmacokinetic parameters and efficacy and safety parameters. RESULTS Twenty-seven eyes from 26 consecutive patients received 66 cycles of SSOAI melphalan and topotecan in combination. All 5 eyes treated as primary therapy responded to the combination chemotherapy and were preserved. Sixteen of the 22 eyes with relapsed or resistant tumors responded, but 3 of them ultimately underwent enucleation at a median of 8 months (range, 7.9-9.1 months). The incidence of grade III and IV neutropenia was 10.6% and 1.5%, respectively, which was comparable with historical controls of single-agent SSOAI melphalan. No episode of fever neutropenia was observed, and no patient required transfusion of blood products. The large variability in melphalan pharmacokinetics was explained by body weight (P <0.05). Concomitant topotecan administration did not influence melphalan pharmacokinetic parameters. There was no effect of the sequence of melphalan and topotecan administration in plasma pharmacokinetics. CONCLUSIONS A regimen combining melphalan and topotecan for SSOAI treatment of retinoblastoma is active and well tolerated. This combination chemotherapy previously showed synergistic pharmacologic activity, and we herein provide evidence of not increasing the hematologic toxicity compared with single-agent melphalan.

Comparison of staging systems for extraocular retinoblastoma: analysis of 533 patients.

IMPORTANCE Different staging systems for extraocular retinoblastoma have been published, but to date they have not been validated in large cohorts. OBJECTIVE To review 533 patients (and pathology slides) with retinoblastoma included in 4 protocols (January 1, 1988, to December 31, 2009) who received uniform treatment. DESIGN AND SETTING Retrospective review in a hospital setting. A critical analysis for detecting inconsistencies and omissions was performed. PARTICIPANTS Patients were reclassified according to the modified St Jude Childrens Research Hospital staging system, Grabowski-Abramson staging system, International Retinoblastoma Staging System (IRSS), and American Joint Committee on Cancer TNM staging system. MAIN OUTCOME AND MEASURE The main outcome measure was disease-free survival (DFS), considering only extraocular relapse as an event. RESULTS In the IRSS and the St Jude system, higher stages correlated with poorer DFS. For intraocular disease, only the TNM system and the IRSS included pathological definitions, and all systems except for the IRSS included substages without differences in DFS. Omissions of factors significantly associated with lower DFS included scleral invasion by the TNM system and massive choroidal invasion by the Grabowski-Abramson system. The St Jude system omits postlaminar optic nerve involvement, but this omission did not correlate significantly with lower DFS because these patients received intensive therapy. No differences in DFS were observed among substages for metastatic disease except for the presence of central nervous system involvement. All staging systems had inconsistencies in definitions of extent of disease. No system provides guidelines for imaging. CONCLUSIONS AND RELEVANCE Only the IRSS and the St Jude system allowed for grouping of patients with increasing risk of extraocular relapse. For lower stages, only the IRSS considers all unequivocal pathological prognostic factors. For higher stages, all systems had redundant information, resulting in an excess of substages.

Detection of minimally disseminated disease in the cerebrospinal fluid of children with high-risk retinoblastoma by reverse transcriptase-polymerase chain reaction for GD2 synthase mRNA.

AIM To evaluate minimally disseminated disease (MDD) in cytologically negative cerebrospinal fluid (CSF) specimens of patients with high-risk retinoblastoma by the detection of the synthase of ganglioside GD2 mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR). METHODS The CSF was evaluated in 26 patients with high risk for CSF relapse: 14 with postlaminar optic nerve invasion, five of them with tumour at the resection margin, five with massive choroidal invasion, three with overt orbital extension and four patients with systemic metastasis. Serial CSF examinations were repeated at different time intervals according to stage and in the event of suspected relapse. GD2 synthase mRNA was evaluated by RT and nested PCR at each procedure. RESULTS MDD was present at diagnosis in six cases (23%) and it was significantly associated to massive optic nerve involvement or history of glaucoma (p<0.05). Three of the children with positive MDD had a CSF relapse. Thirteen patients had negative MDD at diagnosis and one had a CSF relapse. In seven children no ARN could be obtained for PCR analysis and two subsequently relapsed. The probability of CSF relapse was 0.50 (95% confidence interval (CI) 0.13-0.88) for children with MDD and 0.08 (95% CI 0.02-0.46) for those with negative RT-PCR examination of the CSF at diagnosis (p=0.03). CONCLUSIONS MDD in the CSF detected by RT-PCR for GD2-synthase mRNA occurred in 31.7% of evaluable high-risk children with retinoblastoma with no initial central nervous system (CNS) involvement. It was significantly associated to optic nerve involvement and glaucoma and increased risk of CSF relapse.

A phase I study of the anti-idiotype vaccine racotumomab in neuroblastoma and other pediatric refractory malignancies

Pediatric neuroectodermal malignancies express N‐glycolylated gangliosides including N‐glycolyl GM3 (NeuGcGM3) as targets for immunotherapy.

Association of Cone-Rod Homeobox Transcription Factor Messenger RNA With Pediatric Metastatic Retinoblastoma

IMPORTANCE Disseminated retinoblastoma is usually fatal. Identification of small amounts (minimal dissemination [MD]) of tumor cells in extraocular sites might be a tool for designing appropriate treatments. OBJECTIVE To test cone-rod homeobox (CRX) transcription factor as a lineage-specific molecular marker for metastatic retinoblastoma and for evaluation of MD. DESIGN, SETTING, AND PARTICIPANTS In a prospective cohort design study, we evaluated CRX messenger RNA (mRNA) by retrotranscription followed by real-time polymerase chain reaction as a diagnostic test in samples obtained from bone marrow, peripheral blood, and cerebrospinal fluid (CSF) at diagnosis, after induction chemotherapy, and during follow-up. The study was conducted from June 30, 2008, to June 30, 2014. Seventeen retinoblastoma primary tumors, 2 retinoblastoma cell lines, and 47 samples of bone marrow from other cancers (controls) were studied. Seventeen patients with metastatic retinoblastoma (9 at diagnosis, 8 at relapse; age range: 18-41 months) were included. MAIN OUTCOMES AND MEASURES Detection of CRX mRNA as a marker for metastatic retinoblastoma and MD in bone marrow and CSF and its correlation with clinical findings. RESULTS Cone-rod homeobox mRNA was expressed in all tumors (relative expression levels range, 8.1 × 10-5 to 5.6) and cell lines. In control samples, there was no amplification of CRX; only the housekeeping gene (GAPDH) demonstrated amplification. Bone marrow metastatic cells showed expression of CRX mRNA in all 9 children presenting with metastasis at the diagnosis (relative expression levels, 6.0 × 10-5 to 0.67). After induction chemotherapy, no evidence of MD of tumor cells was seen in any of the 8 responding children since only GAPDH showed amplification. In the CSF of children who had a metastatic relapse, CRX mRNA detection was positive in 2 patients in whom no conclusive results were reached by immunocytology for disialoganglioside GD2. Minimal dissemination in the CSF was associated with a clinical relapse in 2 cases. No concomitant MD was evident in the bone marrow in any case. CONCLUSIONS AND RELEVANCE These data suggest that CRX mRNA is a novel marker for retinoblastoma at extraocular sites. In this study among patients with bone marrow metastasis, there was a quick, complete, and sustained molecular response after induction chemotherapy. In all patients with secondary metastasis, CSF relapse occurred independently from the bone marrow, suggesting a sanctuary site.

Immune response to racotumomab in a child with relapsed neuroblastoma.

Immunotherapy targeting ganglioside antigens is a powerful tool for the treatment of high risk neuroblastoma. However, only treatment with anti-GD2 antibodies has been used in clinical practice and other options may be pursued. We report the use of racotumomab, an anti-idiotype vaccine against N-glycolyl neuraminic acid (NeuGc)- containing gangliosides, eliciting an immune response in a child with relapsed neuroblastoma expressing the NeuGcGM3 ganglioside.

Minimal Disseminated Disease in Nonmetastatic Retinoblastoma With High-Risk Pathologic Features and Association With Disease-Free Survival

Importance Fatal metastatic relapse may occur in children with retinoblastoma and high-risk pathologic features (HRPFs). Minimal dissemination (MD) may be an additional tool for risk estimation. The use of cone-rod homeobox (CRX) transcription factor messenger RNA for MD evaluation in metastatic retinoblastoma was previously reported, but no data in nonmetastatic cases with HRPFs are available. Objectives To evaluate whether MD is detectable in patients with nonmetastatic retinoblastoma and to assess its prognostic effect on disease-free survival (DFS). Design, Setting, and Participants This single-institution cohort study of patients with nonmetastatic retinoblastoma and HRPFs used prospectively defined inclusion criteria and a sampling strategy to procure bone marrow (BM) and cerebrospinal fluid (CSF) samples from May 1, 2007, through October 31, 2013. Median follow-up was 38 months (range, 8-89 months). Survival analysis was closed in December 2015, and no further updates were made after that point. Interventions The study evaluated CRX messenger RNA by quantitative polymerase chain reaction in BM and CSF at diagnosis and follow-up. In 14 patients, GD2 synthase was used instead of CRX for CSF evaluation. Patients were treated under uniform guidelines. Main Outcomes and Measures Metastatic relapse. Results The study included 96 children (median age at study inclusion, 26 months; range, 1-168 months; 46 male [47.9%]; 50 female [52.1%]) with nonmetastatic retinoblastoma and HRPFs (isolated massive choroidal invasion in 14, postlaminar optic nerve invasion in 51 [26 with concomitant massive choroidal and 13 with scleral invasion], 12 with scleral invasion without postlaminar optic nerve invasion, and 7 with tumor at the resection margin of the optic nerve) were evaluated at the time of primary or secondary enucleation. Minimal dissemination was detected in 9 patients (7 BM samples and 2 CSF samples) and was associated with extension beyond the resection margin of the optic nerve and scleral involvement, but only the former was independently associated (adjusted odds ratio, 57.0; 95% CI, 4.8-678.2; P = .001). In addition, MD occurred in 8 of the 43 International Intraocular Retinoblastoma Classification group E eyes with glaucoma (18.6%) and in 8 of 80 (10%) and 1 of 16 children (6.3%) who underwent primary or secondary enucleation, respectively. Children with MD had a 3-year DFS of 0.78 compared with 0.98 in those without MD (95% CI for the difference in DFS, 0.17-0.23; P = .004). Conclusions and Relevance These findings identified a high-risk population of children with retinoblastoma and HRPFs with MD. Because the number of events was small, these results, which suggest that children with International Intraocular Retinoblastoma Classification group E retinoblastoma and glaucoma have a higher risk of MD at diagnosis, should not be considered definitive at this time.

Treatment of Nonmetastatic Unilateral Retinoblastoma in Children

Importance Multi-institutional collaborative studies that include large patient populations for the management of retinoblastoma with histopathological risk factors could provide important information for patient management. Objective To evaluate the implementation of a strategy for the management of nonmetastatic unilateral retinoblastoma in children based on standardized diagnostic and treatment criteria. Design, Setting, and Participants This single-arm prospective study applied a strategy based on a single-center experience. The setting was a multicenter study in Latin America (Grupo de America Latina de Oncologia Pediatrica [GALOP]). Participants were children with nonmetastatic unilateral retinoblastoma (staged with the International Retinoblastoma Staging System). The study opened on July 1, 2008, and closed on December 31, 2014. Follow-up was updated until June 30, 2017. Interventions Stage 0 patients (without enucleation) were given conservative therapy without a protocol. Stage I patients (with enucleation and no residual tumor) were divided into a high-risk group (retrolaminar invasion and/or scleral invasion) and a low-risk group (all remaining patients). High-risk children received adjuvant chemotherapy with 4 alternating cycles of regimen 1 (cyclophosphamide [65 mg/kg/d] [plus sodium-2-mercaptoethane sulfonate], idarubicin hydrochloride [10 mg/m2/d], and vincristine sulfate [0.05 mg/kg/d]) and 4 cycles of regimen 2 (carboplatin [500 mg/m2/d, days 1 and 2] and etoposide [100 mg/m2/d, days 1-3]). Low-risk children did not receive adjuvant therapy. Children with buphthalmia received neoadjuvant and adjuvant chemotherapy for a total of 8 cycles. Main Outcomes and Measures Probability of event-free survival (extraocular relapse and death from any cause were considered events). Results Among 187 children registered in the study, 175 were evaluable (92 [52.5%] female; median age, 22 months; age range, 3-100 months). Forty-two were stage 0 children, 84 were stage I low-risk children, and 42 were stage I high-risk children; there were 7 children in the buphthalmia group. With a median follow-up of 46 months, the 3-year probability of event-free survival was 0.97 (95% CI, 0.94-0.99), and the probability of overall survival was 0.98 (95% CI, 0.94-1.00). Stage 0 patients had no events, stage I low-risk patients had 1 event (orbital relapse treated with second-line therapy), stage I high-risk patients had 2 events (1 central nervous system relapse and 1 death from sepsis), and the buphthalmia group had 1 event (orbital relapse, followed by central nervous relapse and death). Conclusions and Relevance Adjuvant therapy may be effective for high-risk unilateral retinoblastoma but is toxic, and neoadjuvant chemotherapy for buphthalmus appears feasible.