Risk estimation of neuroblastoma patients using molecular markers

The pediatric tumor neuroblastoma is a heterogeneous disease: Patients' clinical courses can range from spontaneous regression to fatal progression of the disease. Accordingly, treatment protocols vary from "wait and see" approaches to intensive multimodal therapies. Accurate risk estimation of the patients is therefore mandatory to choose the most adequate therapy. Current trials stratify by a limited number of clinical variables, such as stage of the disease and age of the patient at diagnosis, as well as molecular markers, such as amplification of the oncogene MYCN and loss of the short arm of chromosome 1. However, misclassifications of patients still occur, and thus, a precise prediction of the clinical courses remains a challenge of neuroblastoma research. In recent years, genomic alterations and gene expression profiles of this neoplasm have been characterized thoroughly. It has been shown that the diverse clinical phenotypes are reflected by both specific cytogenetic aberrations and distinct gene expression patterns. Moreover, a variety of DNA copy number changes and gene expression-based classifiers have been described that could predict the outcome of neuroblastoma patients more precisely than established prognostic variables. In this review, the recent advances in the detection and evaluation of molecular prognostic markers for neuroblastoma patients are summarized, and their current and potential contribution to risk stratification systems is discussed.     

The biologic basis for neuroblastoma heterogeneity and risk stratification

PURPOSE OF REVIEW: Neuroblastoma serves as the paradigm for the clinical utility of tumor-specific biologic data for prognostication. This review will describe the genetic and biologic basis for the diverse clinical phenotypes observed in neuroblastoma patients. It will also discuss the current approach to risk classification and how this may change in the future. RECENT FINDINGS: The biologic basis of neuroblastoma has come into clearer focus. PHOX2B is the first bona fide neuroblastoma predisposition gene identified, but is mutated in only a small subset of cases. Somatically acquired alterations at chromosome arms 3p and 11q are highly correlated with acquisition of metastases in the absence of MYCN amplification and may be useful as prognostic markers. The Children's Oncology Group risk classification system has been validated, with current emphasis on further refinement such as reevaluation of the age cutoff used to stratify therapy, and incorporation of additional molecular genetic markers is being studied prospectively. High-throughput genome scale analyses of neuroblastomas are further clarifying the genetic basis of this heterogeneous disease. SUMMARY: Neuroblastoma remains a significant challenge as high-risk patients are treated with intensive multimodal therapies but cure rates remain suboptimal. There is remarkable heterogeneity observed in tumor phenotype, ranging from spontaneous regression to relentless progression. There are literally dozens of clinical and biologic markers that have been proposed as being predictive of disease outcome, but large clinical correlative studies are sharpening the focus of which markers can be used by the clinician to optimize therapy for an individual patient.     

Neuroblastoma in children: Update on clinicopathologic and genetic prognostic factors

Neuroblastoma is the most common extracranial solid tumor in childhood accounting for 8–10% of all childhood malignancies. The tumor is characterized by a spectrum of histopathologic features and a heterogeneous clinical phenotype. Modern multimodality therapy results in variable clinical response ranging from cure in localized tumors to limited response in aggressive metastatic disease. Accurate clinical staging and risk assessment based on clinical, surgical, biologic and pathologic criteria are of pivotal importance in assigning prognosis and planning effective treatment approaches. Numerous studies have analyzed the presence of several clinicopathologic and biologic factors in association with the patient's prognosis and outcome. Although patient's age, tumor stage, histopathologic classification, and MYCN amplification are the most commonly validated prognostic markers, several new gene mutations have been identified in sporadic and familial neuroblastoma cases that show association with an adverse outcome. Novel molecular studies have also added data on chromosomal segmental aberrations in MYCN nonamplified tumors. In this review, we provide an updated summary of the clinical, serologic and genetic prognostic indicators in neuroblastoma including classic factors that have consistently played a role in risk stratification of patients as well as newly discovered biomarkers that may show a potential significance in patients' management.     

Radically different treatment recommendations for newly diagnosed neuroblastoma: pitfalls in assessment of risk

Neuroblastoma risk stratification is based on stage, age, and biology and prescribes surgery for low-risk disease, moderate-dose chemotherapy for intermediate-risk disease, and maximal therapy (including myeloablative treatment with stem cell transplantation) for high-risk disease. Four cases are described that depict pitfalls in risk assessment with potentially far-reaching consequences. This report focuses on a subset of four patients referred for second opinions. Stage was defined by the International Neuroblastoma Staging System. The first recommendations were for maximal therapy, but second opinions were radically different (ie, surgery alone). Ages at diagnosis were 15 to 25 months. Shimada histopathology was unfavorable in three of the four patients, but chromosomal, serum, and urine prognostic markers were favorable. All four patients did well without cytotoxic therapy (follow-up: 2 years 10 months plus to 4 years 8 months plus). Patient 1 had abdominal and upper thoracic/supraclavicular masses (stage 4); the former was resected and the latter spontaneously regressed. Patient 2 had retroperitoneal disease, without bone marrow involvement, but imaging studies showed lesions in vertebral bodies. Biopsies of the latter showed no neuroblastoma and the primary tumor (with regional lymph nodes) was resected, changing stage from 4 to 2B. Patient 3 had a retroperitoneal mass but no distant disease. Though initially deemed to be unresectable, the abdominal tumor was excised, changing the classification from high risk (stage 3 with unfavorable histopathology) to low risk (stage 1). Patient 4 had a pelvic mass, with unfavorable histopathology, and bilateral inguinal lymph node involvement (stage 3); all soft tissue disease was resected. The absence of cortical bone and extensive bone marrow metastatic involvement in a young neuroblastoma patient should cause a shift in attention to biologic prognostic markers. Some patients classified as having high-risk neuroblastoma might actually do well with no cytotoxic therapy.     

Acute tumour lysis syndrome complicating treatment of stage IVS neuroblastoma in infants under six months old

Acute tumour lysis syndrome (ATLS) is a common complication of the treatment of haematopoeitic malignancies. It is also well recognized in many nonhaematopoeitic malignancies of adults. There are very few reports of the syndrome occurring during therapy for the nonhaematopoeitic malignancies of childhood, and none has previously been reported in the treatment of neuroblastoma. We report the cases of four patients presenting to The Hospital for Sick Children (HSC) between 1985 and 1992 who developed ATLS during treatment for stage IVS neuroblastoma. ATLS is a significant risk in patients undergoing therapy for stage IVS neuroblastoma, particularly where this has been delayed. © 1994 Wiley-Liss, Inc.     

Trends of survival in neuroblastoma and independent risk factors for survival at a single institution

To assess the progress of survival in neuroblastoma which varies with many risk factors and to evaluate the influence of these factors on survival as independent risk factors. The study subjects were 159 neuroblastoma patients seen from 1965–1994 at the oldest and largest children's hospital in Japan. Trends of survival in three treatment eras 1965–81, 1982–86, 1987–94 were assessed by the Kaplan-Meier method for different sex, age at diagnosis, the clinical stage, the site of onset, and the histological type. Then the influence on survival of these factors as independent prognostic variables was evaluated by the Cox proportional hazards regression analysis. Age at diagnosis, the clinical stage, the site of onset, the histological type, and the treatment era were independent risk factors in the order of their influence on survival. Unfavorable survival outcomes were obtained for patients with age at diagnosis above 1 year, the clinical stage of VI by the Evans classification, adrenal onset, and neuroblastoma rather than ganglioneuroblastoma. Survival improved from the first to the second and from the second to the third treatment era. Improvement of survival in neuroblastoma took place during the past 3 decades. Age at diagnosis, the clinical stage, and the histological type have still remained overwhelming prognostic factors over the progress in treatment. Med. Pediatr. Oncol. 29:197–205, 1997. © 1997 Wiley-Liss, Inc.     

Cystic Neuroblastoma: Emphasis on Gene Expression, Morphology, and Pathogenesis

Cystic neuroblastoma (CN) is an unusual variant of neuroblastoma characterized by a grossly visible cyst(s) and almost always distinctive microcysts on light microscopy. Rarely, CN will appear solid grossly, but microcystification will be present. We examined the clinical, pathologic, and biologic features of 17 cases of CN. The majority of CN had been detected by prenatal ultrasound. The tumors were favorable stage, stroma-poor, but with low or intermediate mitotic-karyorhectic indices and had favorable biologic markers reflected by aneuploidy and by an absence of N-myc amplification and chromosome 1p deletions. However, the high trk expression typically identified in good risk tumors was absent. Although the complete natural history of CN is not fully defined, our experience suggests that some tumors progress in size, whereas others may spontaneously regress or mature. The clinical outcome is excellent, as is expected in localized and stage 4S neuroblastoma in infancy. Received June 13, 1996; accepted June 19, 1997     

Pneumocystis carinii pneumonia: a late presentation following treatment for stage IV neuroblastoma

This report describes a child who develops Pneumocystis carinii pneumonia 7 months after high-dose chemotherapy for stage IV neuroblastoma. In addition to chemotherapy the child had also been treated with abdominal radiotherapy and 13- cis -retinoic acid. Standard practice has been to treat patients with prophylactic co-trimoxazole for 3 months after high-dose therapy, but this report highlights the intensity and complexity of current treatment for stage IV neuroblastoma and the need to be aware of prolonged lymphopenia after such treatment.     

Prognostic significance of DNA di-tetraploidy in neuroblastoma

BACKGROUND: Identification of biological factors may provide tools to discriminate poor risk neuroblastoma patients of diagnosis, to ultimately offer risk adapted treatment intensity. PROCEDURES: Tumour cell DNA content, MYCN amplification (NMA), deletion of the short arm of chromosome 1 (del 1p) as well as three serological markers were assessed in 179 children with neuroblastoma. RESULTS: Localised regional disease (stage 1 to 3) was diagnosed in 98 patients, and disseminated disease in 81 patients (65 with stage 4, 16 with stage 4s). Median age at diagnosis was 12 months and the median observation time 4 years. Sixty-seven of 179 patients had near di-tetraploid tumours (37%), with a significantly worse prognosis of 44% overall survival at 4 years in comparison with 88% in near triploid tumours (P < .001). The near di-tetraploid group showed a significant correlation with additional adverse biological factors (NMA, del 1p: P < 0.001), age over 1 year (P< 0.001), clinical stage 4 (P< 0.001), elevated ferritin (P = 0.023), and elevated LDH (P< 0.001). Multivariate analysis based on the overall (OS) and event free survival (EFS) estimations revealed that near di-tetraploidy was the most powerful biological factor, with a P-value of <0.001 for EFS and OS, followed by NMA (P = 0.015) for OS and del 1p (P= 0.047) for EFS. CONCLUSIONS: This analysis underlines the important influence of near di-tetraploidy on prognosis, and suggests that more efforts should be undertaken to implement this factor in future studies.     

Age-related profile of neuroblastoma: A comparison of tumors detected by mass-screening with those detected clinically

Infants with neuroblastoma are known to have a favorable prognosis compared to those over 1 year of age. However, there is little biological information about the age-related heterogeneity of neuroblastoma. We evaluated the biological profile comparing cases detected by mass screening with those detected clinically. A total of 238 patients with neuroblastoma were classified into four groups according to their age at diagnosis. Patients in group A were 0–5 months of age (n = 31). Patients in group B were detected clinically and were 6–11 months of age (n = 25). Patients in group C were 6–11 months of age and were detected by mass-screening (n = 97). Patients in group D were more than 12 months of age (n = 85). The age-related heterogeneity was evaluated by Kaplan-Meier survival analysis, several clinical markers (neuron specific enolase, ferritin, vanillylmandelic acid and homovanillic acid) at diagnosis, tumor Ha-ras p21 expression and tumor N-myc amplification. Infant neuroblastoma had unique features in comparison to neuroblastoma diagnosed over 12 months of age. Clinical outcome of the patients in groups A and C was quite favorable. Even patients with stage III or IV disease in group A had a favorable prognosis. However, stage IVs disease in group A was not necessarily associated with a good prognosis and the early death after diagnosis was also characteristic. The biological profile of tumors in group C was similar to that in group A but different from the profile in groups B and D. Tumors in group B had a biological profile intermediate between groups A and D. Cases detected by mass screening (group C) provided a new clinical entity with a good prognosis. The difference in biological profiles might affect their clinical outcome of respective age group. These analyses confirm the significance of prognostic markers and may help to direct an appropriate modality of treatment for the individual patient.     

Circulating tumor DNA in neuroblastoma

As a sympathetic nervous system–derived tumor, aggressive neuroblastoma (NB) is currently attracting interest from researchers seeking diagnostic and prognostic markers via less invasive procedures. The analysis of circulating tumor DNA (ctDNA) in peripheral blood can provide genetic information from multiple tumor lesions and is not dependent on a surgical procedure. The identification of genetic alterations, chromosomal variations, and hypermethylation contained within plasma DNA yields clinical value in the diagnosis, risk stratification, monitoring of treatment effects, and survival prediction for patients. With the widespread application of genome sequencing, droplet digital polymerase chain reaction, and other advanced technologies, the detection of ctDNA may guide therapeutic schedules, enhance the quality of life, and improve the prognosis for patients with NB.     

Neuroblastoma

Neuroblastoma is a heterogeneous disease; tumors can spontaneously regress or mature, or display an aggressive, therapy-resistant phenotype. Increasing evidence indicates that the biological and molecular features of neuroblastoma significantly influence and are highly predictive of clinical behavior. Because of this, neuroblastoma has served as a paradigm for biological risk assessment and treatment assignment. Most current clinical studies of neuroblastoma base therapy and its intensity on a risk stratification that takes into account both clinical and biological variables predictive of relapse. For example, surgery alone offers definitive therapy with excellent outcome for patients with low-risk disease, whereas patients at high risk for disease relapse are treated with intensive multimodality therapy. In this review recent advances in the understanding of the molecular genetic events involved in neuroblastoma pathogenesis are discussed, and how they are impacting the current risk stratification and providing potential targets for new therapeutic approaches for children with neuroblastoma. In addition, the results of significant recent clinical trials for the treatment of neuroblastoma are reviewed.     

Chronic metastatic neuroblastoma

The diversity of neuroblastoma and its clinical course depends on histology, biology and clinical features. We report a male presenting at 4 months of age with an abdominal mass and multiple subcutaneous nodules. The diagnosis was made by histological examination of a subcutaneous nodule and elevated urinary markers. The patient remained well during the subsequent 9 years. During that time no cytostatic treatment was given. Attempt to treat with cis-retinoic acid 10 years later did not result in any significant change of the clinical course. The patient has remained in good clinical condition for a 15-year observation period, having both progressing and regressing distant subcutaneous metastases. Skin nodules are the hallmarks of the indolent clinical course of the disease. We suggest the use of the "chronic neuroblastoma" as a term to describe patients with neuroblastoma showing indolent disease course over a very long period of time, but never achieving complete remission.     

Serum ferritin in stage IV neuroblastoma

Serum ferritin is known to be one of the tumor markers for neuroblastoma. Serum ferritin is elevated in most children with neuroblastoma who are in stages III or IV, but it is not elevated in those in stage I or II. It has also been observed that iron load caused by blood transfusion shows a greater effect on serum ferritin levels than tumor activity due to neuroblastoma. Thus, serum ferritin increases in a linear fashion in children given repeated blood transfusions but the levels increase exponentially in children who have bulky neuroblastomas. Thus, serum ferritin values must be interpreted with caution in patients with advanced stages of neuroblastoma who inevitably require blood transfusions. The authors propose that the pretransfusion and posttransfusion serum ferritin values be in comparison with other tumor markers such as urinary VMA, urinary HVA, and neuron specific enolase. The serum ferritin level should not be used as a sole indicator of tumor activity in neuroblastoma.     

The role of genetic and epigenetic alterations in neuroblastoma disease pathogenesis

Neuroblastoma is a highly heterogeneous tumor accounting for 15 % of all pediatric cancer deaths. Clinical behavior ranges from the spontaneous regression of localized, asymptomatic tumors, as well as metastasized tumors in infants, to rapid progression and resistance to therapy. Genomic amplification of the MYCN oncogene has been used to predict outcome in neuroblastoma for over 30 years, however, recent methodological advances including miRNA and mRNA profiling, comparative genomic hybridization (array-CGH), and whole-genome sequencing have enabled the detailed analysis of the neuroblastoma genome, leading to the identification of new prognostic markers and better patient stratification. In this review, we will describe the main genetic factors responsible for these diverse clinical phenotypes in neuroblastoma, the chronology of their discovery, and the impact on patient prognosis.     

Premature physeal closure following 13‐cis‐retinoic acid and prolonged fenretinide administration in neuroblastoma

Retinoid therapy has contributed to improved outcomes in neuroblastoma. Clinical trials of fenretinide report favorable toxicity and disease stabilization in patients with high risk (HR) neuroblastoma. Skeletal effects have been described with other retinoids, but not with fenretinide to date. Two patients with HR, metastatic, refractory neuroblastoma received protracted courses of oral fenretinide for more than 5 years’ duration. Both developed premature long bone physeal closure, causing limb length discrepancies; their neuroblastoma remains in remission. The radiographic and clinical findings reported suggest these skeletal abnormalities may be a consequence of treatment with 13‐cis‐retinoic acid (13cisRA) followed by prolonged oral fenretinide exposure.     

Opsoclonus-myoclonus-ataxia syndrome in neuroblastoma: clinical outcome and antineuronal antibodies-a report from the Children's Cancer Group Study

BACKGROUND: Opsoclonus-myoclonus-ataxia (OMA) is a paraneoplastic neurologic syndrome affecting 2-3% of children with neuroblastoma. Although children with OMA and neuroblastoma may have higher survival, many experience a significant amount of late neurologic impairment, which may be immunologically mediated. The aim of this study was to compare the outcome of neuroblastoma patients with and without OMA, relating to prognostic factors, treatment, and the presence or absence of anti-neuronal antibodies. PROCEDURE: Questionnaires were mailed out requesting information on the current neurologic status of patients who submitted sera at diagnosis to the Children's Cancer Group serum bank from 1980 to 1994. Information was requested on clinical and biological patient characteristics as well as clinical aspects of the patients identified as having OMA syndrome, including presentation and treatment for OMA, late sequelae of OMA, the presence or absence of antineuronal antibodies, and survival. Sera from 16 of the OMA patients and 48 case-controls with neuroblastoma were assayed for anti-neuronal antibodies. RESULTS: Of the 675 responses received, 21 patients had OMA. Ninety percent of OMA patients presented with non-metastatic disease, vs. 35% of non-OMA patients. Estimated 3-year survival for the OMA patients with nonmetastatic disease (stage I, II, III) greater than 1 year of age was 100% vs. 77% for similar non-OMA patients (P = 0.0222). At follow-up, 14/19 evaluable OMA patients displayed some form of developmental or neurologic abnormality. There was no significant correlation of late sequelae with antineuronal antibodies, age, time between OMA symptoms and diagnosis, or treatment given for tumor or OMA. There was a significant correlation of late sequelae with lower stage disease (I and II) compared to more advanced disease (III and IV). CONCLUSIONS: Patients with OMA and neuroblastoma have excellent survival but a high risk of neurologic sequelae. Favorable disease stage correlates with a higher risk for development of neurologic sequelae. The role of anti-neuronal antibodies in late sequelae of OMA needs further clarification.     

Neuroblastoma: the case for screening infants in North America

Neuroblastoma is a disease in which easily measured tumor markers are excreted. It is curable when diagnosed in an early stage and at an early age, and it has a high incidence relative to other serious childhood diseases. Recent advances in screening infants for neuroblastoma by detection of urinary homovanillic acid and vanillylmandelic acid, the most useful markers of neuroblastoma, are described. Based on results from Japan's mass-screening program and on the authors' observations, it is estimated that mass screening for neuroblastoma could save 260 lives annually in the United States.     

Liver involvement in neuroblastoma: the Memorial Sloan-Kettering Experience supports treatment reduction in young patients

BACKGROUND: We reviewed clinical and biologic findings in a series of infants with neuroblastoma (NB) in liver. The aim was to gain insights into improving therapy. PATIENTS AND METHODS: Among 19 newly or recently diagnosed infants with NB in liver, 1987-2002, those with stage 4 involving bone received chemotherapy, while those without bone or extensive bone marrow (BM) involvement were observed or received limited treatment if NB caused life-threatening symptoms. We assessed results in the context of NB treatment risk stratification, which is based on age, stage, and selected biologic features (MYCN, ploidy, histology). RESULTS: Six of eight infants with bone involvement became long-term event-free survivors including 1/2 with MYCN amplification and four who received only 4-6 cycles of chemotherapy; at the end of treatment, four infants had abnormalities in liver +/- the primary site, but these resolved. All 11 infants without bone lesions became long-term survivors with either no cytotoxic therapy or only one cycle of chemotherapy (+/- radiotherapy to liver), including four who had stage 4 and one stage 4S patient who still had NB in BM at age 15 months. CONCLUSIONS: Treatment reduction should be considered for subsets of infants with non-MYCN-amplified widespread NB: stage 4 without bone or extensive BM involvement may not require cytotoxic therapy, stage 4S with symptomatic hepatomegaly may not require multiple cycles of chemotherapy, and classic stage 4 may do well with limited chemotherapy. Persistent liver abnormalities post-treatment may not require continued therapy to achieve a radiologic complete remission.     

Incidental neuroblastoma

The diagnosis of neuroblastoma in its early stage, especially in asymptomatic children, with the so-called incidentally diagnosed disease, may be associated with a good prognosis. The aim of this study is an attempt at analyzing this problem. Between 1 January 1993 and 30 April 1998, 40 children with newly diagnosed neuroblastoma started therapy at the authors' department. The disease was diagnosed incidentally in 5 (12.5%) patients. In no incidentally diagnosed child was stage IV disease detected, while in the remaining patients its incidence was 71%. All the children (median age 2 months) with incidental diagnosis have remained alive (median 39 months) in continuos remission without treatment. Among 35 children (median age 2 years and 7 months) with overt neuroblastoma, 18 died (median survival time 14.5 months). Seventeen patients have remained alive (median 45 months). The results show that children with incidentally diagnosed neuroblastoma are characterized by a more favorable prognosis than children with clinical disease.