Neuroblastoma

Introduction

Neuroblastoma is the most common solid extracranial malignancy of childhood and the most common malignant tumor in infants. The overall incidence of neuroblastoma is 10.2 per million children younger than 15 years of age. Progressive or relapsed neuroblastoma is responsible for approximately 15% of all childhood cancer deaths. Neuroblastoma is rarely encountered in children older than 10 years of age and has a poor prognosis in older children. The incidence of neuroblastoma is higher in developed countries when compared to developing countries, but specific environmental agents contributing to neuroblastoma development are poorly understood. The lower incidence in developing countries may be due to early death from progressive disease prior to diagnosis. The clinical behavior of neuroblastoma is quite heterogeneous, with a spectrum that ranges from tumors that spontaneously regress or mature to tumors with a very aggressive, malignant phenotype. Progress in the understanding of neuroblastoma biology and risk-stratification over the last 50 years has led to risk-stratification of the likelihood for disease recurrence and treatment paradigms that emphasize therapy reduction for patients with low- and intermediate-risk disease and intensification of multimodality therapy for patients with high-risk disease.

Pathology

The German pathologist Rudolph Virchow is generally credited with being the first to describe the histologic appearance of what is now known as neuroblastoma in his 1864 article entitled, “Hyperplasia of the Pineal and Suprarenal Glands.” The first to use the term neuroblastoma was James Homer Wright, who, in 1910, described the classic appearance of rosettes of tumor cells around central neural fibrils and noted the similarity to the morphology of the fetal adrenal gland. Wright also noted the association between the common sites of tumor development and the pattern of migration of primitive neural cells. Indeed, neuroblastoma is an embryonal tumor of the sympathetic nervous system that is thought to originate during fetal or early postnatal life in cells derived from the neural crest. Therefore, the anatomic location of neuroblastoma is variable and can originate anywhere along the path that neural crest cells migrate, including the adrenal medulla (abdomen), paraspinal sympathetic ganglia (neck, chest, abdomen) or the sympathetic paraganglia such as the organ of Zuckerkandl (pelvis). The abdomen is the most common site for neuroblastoma (75% of tumors), with the adrenal medulla being the most common site of origin among abdominal tumors ( Fig. 63.1 ). Thoracic tumor location is associated with better prognosis, while adrenal tumor location is associated with an inferior prognosis when controlling for MYCN amplification, age, stage, and tumor histology.

A medical diagram shows neuroblastoma locations with percentage distribution across cervical, thoracic, retroperitoneal, and pelvic regions. The medical diagram shows the anatomical distribution of neuroblastoma in a pediatric body, highlighting five major regions where the tumor commonly arises, along with their relative frequency. At the top, the cervical region is labeled with a small tumor near the neck and marked as one percent of cases. In the thoracic region, a posterior mediastinal neuroblastoma is shown adjacent to the spine and heart, representing twenty percent of cases. On the left side of the retroperitoneum, a paraspinal ganglion origin neuroblastoma is depicted, accounting for twenty five percent of cases. On the right side, a retroperitoneal neuroblastoma of adrenal medulla origin is shown near the kidney and adrenal gland, representing fifty percent of all neuroblastoma cases, making it the most common location. Finally, a small tumor in the pelvic region is labeled as pelvic neuroblastoma, accounting for four percent of cases. — Fig. 63.1

Neuroblastoma is one of the small, round blue cell tumors of infancy or childhood. Younger patient age and increased histologic differentiation are associated with improved prognosis in neuroblastoma patients. Overall, neuroblastoma is the least differentiated and most malignant of a family of neuroblastic tumors that includes ganglioneuroblastoma (larger, intermediately differentiated cells) and ganglioneuroma (highly differentiated, benign cells; Fig. 63.2 ). More specifically, neuroblastoma can be distinguished histologically by the presence of neuritic processes (neuropil) and Homer Wright rosettes (neuroblasts surrounding eosinophilic neuropil). Scattered ganglion cells or immature chromaffin cells can also be seen. The histologic appearance of neuroblastic tumors may range from undifferentiated cells to fully mature ganglion cells. In addition, neuroblastic tumors have variable degrees of Schwannian cell stroma, reactive nonneoplastic tissue recruited by the tumor cells. ^,

A panel of histologic views shows stages of neuroblastoma differentiation from undifferentiated to ganglioneuroma with insets highlighting cellular features. The panel of histologic views shows four labeled histological panels A through D, each representing a different stage in the neuroblastoma to ganglioneuroma spectrum under hematoxylin and eosin staining. Panel A shows an undifferentiated neuroblastoma composed of densely packed small round cells with high nuclear to cytoplasmic ratios, minimal stroma, and areas of necrosis. The inset highlights classic features such as Homer Wright rosettes indicated by arrows and mitotic figures marked by a bar. Panel B shows a differentiating neuroblastoma with larger tumor cells showing abundant eosinophilic cytoplasm and primitive ganglion like cells. The inset demonstrates differentiating cells with prominent nucleoli marked by arrowheads and neurofibrillary matrix indicated by a arrow. Panel C shows a ganglioneuroblastoma with loosely arranged Schwannian stroma and islands of differentiating neuroblasts. The architecture is less cellular with spindle shaped cells in a more fibrillary matrix. Panel D shows a mature ganglioneuroma with a uniform spindle cell background and scattered mature ganglion cells, one of which is marked by a arrow. The background is composed of Schwannian stroma to benign ganglioneuroma. — Fig. 63.2

By definition, neuroblastoma is Schwannian stroma-poor, while the more differentiated neuroblastic tumors ganglioneuroblastoma and ganglioneuroma have a higher proportion of Schwannian stroma. Neuroblastoma can be further characterized as having favorable or unfavorable histology, which is dependent on patient age, histologic differentiation status, and a marker of cell proliferation and division known as the mitosis-karyorrhexis index (MKI; Fig. 63.2 ). ^, Accordingly, the age and histology-based International Neuroblastoma Pathologic Classification System (INPC; Shimada system; Table 63.1 ) classifies neuroblastic tumors into three morphologic categories (i.e., neuroblastoma, ganglioneuroblastoma, and ganglioneuroma) and contributes to contemporary risk-stratification of neuroblastoma patients, which is discussed in the following. ^,

Table 63.1

Prognostic Evaluation of Neuroblastic Tumors According to the International Neuroblastoma Pathology Classification (INPC/Shimada System)

Adapted from Shimada H, Ambros IM, Dehner LP, et al. The international AIB pathology classification (the Shimada System). Cancer . 1999;86:364–372.

International Neuroblastoma Pathology Classification	Histologic Characteristics	Prognostic Group
Neuroblastoma	Schwannian stroma-poor
<1.5 years	Poorly differentiated or differentiating & low or intermediate MKI tumor	Favorable
1.5–5 years	Differentiating & low MKI tumor	Favorable
<1.5 years	a) Undifferentiated tumor b) High MKI tumor	Unfavorable
1.5–5 years	a) Undifferentiated or poorly differentiated tumor b) Intermediate or high MKI tumor	Unfavorable
≥5 years	All tumors	Unfavorable
Ganglioneuroblastoma, intermixed	Schwannian stroma-rich	Favorable
Ganglioneuroblastoma, nodular	Composite Schwannian stroma-rich/stroma-dominant and stroma-poor	Unfavorable
Ganglioneuroma	Schwannian stroma-dominant	Favorablea
Maturing
Mature

MKI , mitotic karyorrhexis index.

Clinical Presentation

Patients with neuroblastoma usually present with signs and symptoms that reflect the primary site and extent of disease, although localized disease is often asymptomatic. As 75% of neuroblastoma occurs in the abdominal cavity, abdominal pain or an abdominal mass detected on physical examination are common clinical features. Other primary sites of neuroblastoma include the posterior mediastinum (20%), neck (1%), and pelvis (4%; Fig. 63.1 ). Altered defecation or urination can be caused by mechanical compression from a pelvic tumor or by spinal cord compression from a paraspinal tumor. Spinal cord compression may also manifest as an altered gait or new-onset lower extremity weakness or paralysis. Respiratory distress or dysphagia can be caused by thoracic tumors. A tumor in the neck or upper thorax can also produce Horner syndrome (ptosis, miosis, anhidrosis), enophthalmos, and heterochromia of the iris. A striking, immune-mediated paraneoplastic syndrome called opsoclonus-myoclonus-ataxia syndrome (OMAS) can be triggered by neuroblastoma and is characterized by rapid, involuntary eye movements (opsoclonus), brief involuntary twitching of muscle (myoclonus), gait disturbances, irritability, insomnia, nausea, vomiting, and speech difficulty. Two-thirds of neuroblastoma patients with OMAS have thoracic tumors and most have tumors of a modest volume, presumably due to early presentation given the clinical symptoms. A recent randomized, prospective clinical trial conducted by the Children’s Oncology Group (COG ANBL00P3) demonstrated that the addition of intravenous immunoglobulin (IVIG) to prednisone and risk-adapted chemotherapy improves the response rate (80.8%) compared to OMAS patients who did not receive IVIG (40.7%; P = .0029). Although the oncologic outcome for these patients is generally very good, they often have significant long-term neurologic sequelae of their disease. Additional signs and symptoms that reflect excessive catecholamine or vasoactive intestinal polypeptide secretion in a minority of neuroblastomas include diarrhea, weight loss, and hypertension.

More than 60% of patients with neuroblastoma have metastatic disease at diagnosis ( Fig. 63.3 ). These patients are often ill-appearing due to systemic signs and symptoms caused by widespread disease. Neuroblastoma most commonly metastasizes to the bone marrow, lymph nodes, and cortical bone. The brain, spinal cord, heart, and lungs are rare sites of metastases, except with end-stage disease. A specific pattern of widespread metastases to the liver, skin, and limited involvement of the bone marrow (<10% of marrow cells), but without bone metastases, in infants and children less than 18 months old is characteristic of stage MS disease that is associated with a generally favorable prognosis and discussed in greater detail later in the chapter. Metastatic disease may also be associated with darkened areas around the eyes referred to as “raccoon eyes,” because of retroorbital venous plexus spread ( Fig. 63.3 ). This is an ominous physical sign, as is the presence of a limp in children without a history of head or extremity trauma.

A composite of clinical and diagnostic views shows neuroblastoma with skeletal metastases, orbital involvement, abdominal tumor, and cytology smear. The composite of clinical and diagnostic views shows six diagnostic and clinical panels labeled A through E that illustrate the presentation and spread of neuroblastoma in a pediatric patient. Panel A includes anterior and posterior views of a whole body nuclear medicine scan showing intense radiotracer uptake in multiple areas, especially the skull and long bones, marked by arrows. Panel B shows a coronal computed tomographic scan of the abdomen, where a large retroperitoneal mass is evident adjacent to the kidneys; arrowheads indicate affected regions. Panel C shows an axial computed tomographic scan of the orbit revealing bony destruction of the orbital walls, indicated by arrowheads, representing metastatic spread to the bone. Panel D is a clinical photograph of a child's face showing periorbital ecchymosis or raccoon eyes. Panel E presents a bone marrow aspirate smear stained to highlight clusters of neuroblastoma cells with characteristic features such as large nuclei and dense chromatin, forming rosettes. — Fig. 63.3

Diagnosis

The initial diagnostic workup of neuroblastoma is typically characterized by detection of a mass by physical examination, plain radiography (especially if the mass has calcifications), or ultrasound. The workup proceeds with confirmation of the mass and delineation of its anatomic details by cross-sectional imaging, detection of elevated urinary catecholamines, a workup for metastatic disease, and ultimately histopathologic and molecular examination of tumor tissue from primary or metastatic sites.

Primary Site Diagnostic Imaging

Standard x-rays can be a useful tool for demonstrating the presence of a posterior mediastinal mass, which in an infant or young child is usually a thoracic neuroblastoma. A Pediatric Oncology Group (POG) study demonstrated that a mediastinal mass was discovered incidentally by chest radiograph in almost half of patients who were ultimately diagnosed with thoracic neuroblastoma. Abdominal radiography is less often the modality by which a neuroblastoma is discovered. However, as many as half of abdominal neuroblastomas are detectable as a mass with fine calcification by standard x-rays. Ultrasonography (US) is most often used during the initial assessment of a suspected abdominal mass but does not typically yield the necessary anatomic definition used in definitive imaging assessment of neuroblastoma or its staging.

Cross-sectional imaging performed with computed tomography (CT) or magnetic resonance imaging (MRI) is used to evaluate the detailed anatomy of primary site neuroblastoma. CT scan can demonstrate calcification in almost 85% of neuroblastomas. Cross-sectional imaging can be used to facilitate differentiation of primary adrenal neuroblastoma from renal tumors because neuroblastomas contain calcifications, often cross the abdominal midline, and encase blood vessels with much greater frequency than pediatric renal tumors. Furthermore, adrenal neuroblastomas do not typically produce the “claw-sign” characteristic of renal tumors. Intraspinal extension of paraspinal neuroblastomas in the thorax or abdomen is frequently detected by CT scan and more precisely evaluated using MRI. CT and MRI are also used to document the multitude of imaging-defined risk factors that are critical in the contemporary staging and risk-stratification of neuroblastoma, as discussed later.

Catecholamine Metabolites and Metaiodobenzylguanidine Imaging

Neuroblastoma is characterized by the relatively unique capacity for secretion of catecholamine products, the metabolites of which can be detected in the urine of more than 90% of patients with neuroblastoma and leveraged for diagnostic confirmation. Thus, evaluation of a urine specimen for elevated homovanillic acid (HVA) and vanillylmandelic acid (VMA) is mandatory in the workup of all patients with suspected neuroblastoma. Random urine samples are preferable to 24-hour urine estimations for younger children. Documentation of elevated urinary HVA/VMA is required if the diagnosis of neuroblastoma is being made solely by the identification of neuroblasts in the bone marrow. Urinary levels of HVA/VMA can also be used as markers of tumor progression to guide surgical decision making in patients with low-risk neuroblastoma being managed with observation only or for surveilling for tumor relapse.

Metaiodobenzylguanidine (MIBG) is a chemical analog of the catecholamine neurotransmitter norepinephrine. Therefore, MIBG localizes to adrenergic tissues including neuroblastoma at its primary and metastatic sites. Approximately 90% of neuroblastomas are MIBG-avid. MIBG scintigraphy is the preferred imaging study to determine metastatic bone and bone marrow involvement by neuroblastoma, having largely replaced technetium-99m methylene diphosphonate ( ^99m Tc-MDP) bone scans ( Fig. 63.3 ). MIBG is therefore a critical imaging modality in the diagnostic confirmation of primary and metastatic neuroblastoma. In those patients with non–MIBG-avid neuroblastoma, PET-CT is often performed for identification of metastatic disease. The selective uptake of MIBG by neuroblastoma cells has been leveraged for therapeutic intervention using ¹³¹ I-MIBG, which has been demonstrated to be safe, feasible, and potentially effective for both relapsed and newly diagnosed neuroblastoma in clinical trials.

Bone Marrow Examination

Bone marrow examination by both trephine biopsy and aspiration is another important component of the neuroblastoma metastatic workup ( Fig. 63.3 ), as the bone marrow is a common site of tumor spread. To collect more accurate information, taking specimens from multiple sites is recommended. Immunohistochemical staining of obtained bone marrow with antibodies such as antiganglioside G _D2, S-100, neuron-specific enolase, and ferritin is also useful to help reduce the number of false-negative cases.

Tumor Staging

As previously mentioned, one of the notable characteristics of neuroblastoma is the substantial heterogeneity of the disease, which ranges from spontaneous regression or maturation, even without therapy, to a highly malignant, aggressive phenotype that is poorly responsive to current intensive, multimodal therapy. Increasing evidence indicates that the biologic and molecular features of neuroblastoma are highly predictive of clinical behavior. Therefore, neuroblastoma has served as a paradigm for phenotypic risk assessment and treatment assignment whereby those at high risk for disease relapse are given intensive multimodal therapy. Those at low risk for relapse can have treatment intensity diminished to avoid therapy-associated toxicity while still achieving a high rate of cure. The predictive value of these biologic factors is important not only for the oncologist when considering appropriate chemotherapy, but also for the surgeon when considering the timing and extent of an operative resection in a child with neuroblastoma. Risk-stratification starts with radiographically based tumor staging, and then proceeds to incorporate a multitude of factors including patient age, tumor histology, and molecular genetic information.

Tumor Staging—L1, L2, M, or MS

Contemporary staging for neuroblastoma is performed using the International Neuroblastoma Risk Group Staging (INRG) Staging System (INRGSS; Table 63.2 ). In the INRGSS, localized tumors are designated L1 or L2 depending on the absence or presence of one or more of 20 image-defined risk factors (IDRFs) across multiple organ systems. These IDRFs are shown in Table 63.3 and generally reflect encasement of vital structures, primarily vessels and nerves, as determined by diagnostic imaging studies. Pertaining to arterial encasement, an IDRF is present if the tumor encases greater than 50% of the circumference of the artery. For venous encasement, an IDRF is present when greater than 50% of the circumference of the vein is encased or if there is collapse of the vessel with no visible flow on contrasted imaging ( Fig. 63.4 ). The INRGSS was most recently assessed and validated by the INRG in 2009 using a cohort of 661 patients treated in Europe. In this study, 261 patients with INRGSS stage L2 disease exhibited lower 5-year event-free survival (EFS) than 213 patients with L1 disease (78% ± standard error [SE] 4% vs. 90% ± SE 3%; log-rank P = .0010). The 5-year overall survival (OS) was also lower for those with L2 disease compared to L1 (89% ± SE 3% vs. 96% ± SE 2%; P = .0068). In addition, the presence of IDRFs was previously shown to predict completeness of surgical resection and to be associated with the frequency of surgical complications. ^, The prior neuroblastoma staging system (International Neuroblastoma Staging System—INSS), a surgicopathologic staging system, may continue to be important for interpretation of the historic literature and contemporary literature reporting long-term outcomes in patients who were treated according to this staging system.

Table 63.2

International Neuroblastoma Risk Group Staging System (INRGSS)

Adapted from Monclair T, et al. The International Neuroblastoma Risk Group (INRG) staging system: an INRG Task Force report. J Clin Oncol . 2009;27:298–303.

Stage	Description
L1	Localized tumor not involving vital structures, as defined by the list of image-defined risk factors, and confined to one body compartment.
L2	Locoregional tumor with presence of one or more image-defined risk factors.
M	Distant metastatic disease (except stage MS tumor).
MS	Metastatic disease in children younger than 18 months, with metastases confined to skin, liver, and/or bone marrow.

Table 63.3

Neuroblastoma Image-Defined Risk Factors (IDRF)

Ipsilateral Tumor Extension Within Two Body Compartments
Neck-chest, chest-abdomen, abdomen-pelvis
Neck
Tumor encasing carotid and/or vertebral artery and/or internal jugular vein
Tumor extending to base of skull
Tumor compressing the trachea
Cervicothoracic junction
Tumor encasing brachial plexus roots
Tumor encasing subclavian vessels and/or vertebral and/or carotid artery
Tumor compressing the trachea
Thorax
Tumor encasing the aorta and/or major branches
Tumor compressing the trachea and/or principal bronchi
Lower mediastinal tumor, infiltrating the costovertebral junction between T9 and T12
Thoracoabdominal
Tumor encasing the aorta and/or vena cava
Abdomen/pelvis
Tumor infiltrating the porta hepatis and/or the hepatoduodenal ligament
Tumor encasing the origin of the celiac axis and/or the superior mesenteric artery
Tumor invading one or both renal pedicles
Tumor encasing the aorta and/or vena cava
Tumor encasing the iliac vessels
Pelvic tumor crossing the sciatic notch
Intraspinal tumor extension of any portion provided that:
More than one-third the spinal canal in axial plane is invaded and/or perimedullary leptomeningeal spaces are not visible and/or spinal cord signal is abnormal
Infiltration of adjacent organs/structures
Pericardium, diaphragm, kidney, liver, duodenopancreatic block, and mesentery
*Conditions to be recorded, but not* considered IDRFs**
Multifocal primary tumors
Pleural effusion, with or without malignant cells
Ascites, with or without malignant cells

Adapted from Monclair T. et al. The International Neuroblastoma Risk Group (INRG) staging system: an INRG Task Force report. J Clin Oncol . 2009;27:298–303.

A diagram shows vascular involvement by a tumor with and without defined risk factors based on the degree of encasement. The diagram shows two side by side schematic comparisons illustrating tumor interaction with nearby blood vessels and the presence or absence of defined risk factors I D R F. s. in pediatric abdominal tumors. The left panel is labeled No I D R F and shows a tumor with less than fifty percent circumferential contact with a nearby artery and a slightly compressed vein that still retains a visible lumen. This configuration is interpreted as minimal vascular involvement with no critical risk. The right panel is labeled plus I D R F and demonstrates a tumor with more than fifty percent contact with the artery, described as partial encasement, and the vein is shown as completely flattened with no visible lumen, indicating total encasement. Arrows point to specific interaction types with descriptive annotations indicating how contact percentage and loss of vascular lumen define I D R F presence. — Fig. 63.4

Metastatic tumors are defined as stage M, except for stage MS, which is defined by a specific pattern of widespread metastases to the liver, skin, and limited involvement of the bone marrow (<10% of marrow cells) in infants and children less than 18 months old. These patients cannot have cortical bone involvement and still be considered stage MS. For patients with INRG stage M or MS disease, a description of primary tumor IDRFs may still be helpful to plan eventual primary tumor surgery and to ascertain the rate of surgical complications, although IDRFs do not help define stage in patients with metastatic disease.

In 1971, D’Angio, Evans, and Koop reported a number of patients with a “special” variant of metastatic neuroblastoma, termed 4S (now referred to as MS in the INRGSS). These patients are infants who typically had a single, small primary tumor but had extensive metastatic disease in the liver, skin nodules (“blueberry muffin” lesions), and small amounts of disease in the bone marrow (<10% of the mononuclear cells). Patients with MS neuroblastoma are quite remarkable because the large amount of disease can undergo spontaneous regression, even without treatment, and the infants ultimately have no evidence of disease. In the past, only supportive therapy had been recommended for MS neuroblastoma because of the high incidence of spontaneous regression and the good prognosis. Most of these patients have a tumor with favorable biology ( MYCN non-amplified, favorable INPC/Shimada histology, and hyperdiploidy/DNA index >1). Therefore, they are assigned to the low-risk classification and receive no therapy. However, despite the generally benign course of their malignancy, these infants can die of complications caused by the initial bulk of their disease. Limited chemotherapy, local irradiation, or minimal resection can be used to treat infants with life-threatening symptoms of hepatomegaly. Decompressive laparotomy with creation of a Silastic pouch may be needed for those with significant hepatomegaly that causes either respiratory compromise secondary to diaphragmatic elevation or obstruction of the inferior vena cava. This procedure may help avoid life-threatening events until shrinkage of the liver is achieved by either spontaneous regression or therapy. The COG ANBL1232 protocol discussed below assessed the treatment of patients with MS neuroblastoma according to patient age, symptoms, tumor biologic features, and response-based characteristics.

While not a component of pretreatment staging or risk-stratification, how IDRFs or tumor volume change during neoadjuvant systemic therapy is a relevant question for surgical timing and planning. In a cohort of 107 patients with neuroblastoma and IDRFs, it was demonstrated that MYCN -amplified and histologically undifferentiated neuroblastomas had a greater number of mean IDRFs than nonamplified and more differentiated tumors. This analysis also suggested that both the number of IDRFs and the tumor volume were likely to decline more in MYCN -amplified and less differentiated tumors with neoadjuvant chemotherapy. In a cohort of 88 patients treated with neoadjuvant chemotherapy for high-risk neuroblastoma, a reduction in the number of IDRFs was demonstrated in 81.8% of the patients, with a mean (±standard deviation) decrease of 2.9 (±2.5) IDRFs per patient. Intuitively, reduction in IDRFs correlated strongly with volumetric tumor reduction in this analysis. This group also previously showed that MYCN-amplification was associated with greater reduction in primary tumor volume in response to neoadjuvant chemotherapy for high-risk neuroblastoma patients. In summary, although high-risk neuroblastoma, MYCN amplification, and undifferentiated tumor histology are associated with a poorer prognosis, a significant reduction in IDRFs and tumor volume can be anticipated in these patient groups in response to neoadjuvant chemotherapy. These findings reinforce the importance of delayed rather than upfront surgery in these groups. How specific IDRFs change with neoadjuvant chemotherapy and how specific IDRFs may associate with oncologic outcomes are subjects of future interest in neuroblastoma surgery.

Histopathologic Confirmation

Because L1 tumors do not have IDRFs, they should either be completely resected or observed without surgical intervention. In contrast, L2, M, and MS tumors should generally undergo biopsy at diagnosis. Most COG protocols recommend 1 cm ³ of tissue for histopathologic confirmation, completion of necessary molecular studies, and biobanking of tumor specimens for protocol-driven or future scientific translational research. Traditionally, this volume of tissue has been most reliably obtained using an open or minimally invasive surgical biopsy. However, a multiinstitutional retrospective study including 243 patients with neuroblastoma compared open incisional biopsy to percutaneous core-needle biopsy and found that core biopsy was sufficient for diagnosis and determination of MYCN oncogene status in patients with neuroblastoma. These authors concluded that immediate tissue assessment by a pathologist to determine specimen quality and obtaining multiple cores were critical components of this procedure to ensure adequate tissue for complete molecular assessment. Determining evidence-based percutaneous biopsy guidelines and associated outcomes are aims of future neuroblastoma research, especially with increasing identification of biomarkers that guide therapy but require sufficient tissue to document, such as ALK aberrations.

Molecular Biology

Amplification of the MYCN oncogene is the most recognized driver of high-risk neuroblastoma and is present in approximately 20% of neuroblastoma cases overall. Clinically, MYCN amplification is typically ascertained by fluorescence in situ hybridization and results in assignment of high-risk disease in most cases ( Fig. 63.5 ). ^, The MYCN copy number, which can range from 5- to 500-fold amplification, is usually consistent among primary and metastatic sites and at different times during tumor evolution and treatment. Transgenic overexpression of MYCN results in neuroblastoma in murine and zebrafish models. Taken together, these findings demonstrate that MYCN amplification is not only a biomarker associated with high-risk disease, but it is also a fundamental driver of malignant transformation and progression in sympathetic neuroblasts. In addition, other MYC family members have been implicated in neuroblastoma pathogenesis. Focal amplification of distal enhancers (DNA-regulatory elements that activate transcription of a gene) or hijacking of enhancer elements by chromosomal translocation drive increased MYC expression (which codes for the c-MYC protein) in high-risk neuroblastoma.

A fluorescent in situ hybridization illustration shows normal chromosome spread in panel A and M Y C N gene amplification with extrachromosomal elements in panel B. The fluorescent in situ hybridization illustration shows panels labeled A and B, highlighting M Y C N gene status in chromosomes. Panel A depicts a normal metaphase chromosome spread with numerous compact, stained chromosomes, each containing scattered fluorescent signals, representing two M Y C N gene copies per chromosome set. Panel B displays a chromosome spread from a tumor cell exhibiting M Y C N amplification. The chromosomes appear more loosely organized, and multiple bright signals cluster within a large extrachromosomal body labeled with an asterisk, indicating the presence of double minutes extrachromosomal D N A segments associated with oncogene amplification. Arrows point to abnormal signal accumulations and chromosomal structures, and a dashed arrow highlights a segment with homogeneously staining regions, suggesting intrachromosomal amplification. — Fig. 63.5

Gain-of-function pathogenic somatic variants in the ALK receptor tyrosine kinase oncogene are the most common single-nucleotide variants found in neuroblastoma. Cooperative aberrations in ALK and MYCN are frequent and play a role in neuroblastoma pathogenesis and progression. ^, In addition, inherited germline pathogenic variants in ALK are responsible for most familial neuroblastoma cohorts. Multiple studies have demonstrated an increased frequency of ALK pathogenic variants when matched primary and relapsed neuroblastomas were compared, implicating expansion of subclonal ALK mutations found at diagnosis in neuroblastoma disease relapse. ^, ALK is amenable to pharmacologic inhibition by small molecules including crizotinib; however, the eventual evolution of therapeutic resistance is common. ^, Lorlatinib, an inhibitor of ALK and ROS1, may overcome this therapeutic resistance in ALK-driven refractory/relapsed neuroblastoma; however, evolution of lorlatinib resistance is also a barrier to durable treatment success. ^, The COG ANBL1531 high-risk neuroblastoma protocol assigned patients with tumors containing ALK aberrations to receive crizotinib during induction therapy and throughout treatment.

Abnormalities in chromosomal copy number profiles play a role in neuroblastoma risk-stratification. Normal human somatic cells have a diploid chromosomal complement of 46 chromosomes, or two copies of each of 23 chromosomes. The majority (55%) of primary neuroblastomas are triploid or “near-triploid/hyperdiploid” and contain between 58 and 80 chromosomes; the remainder (45%) are either near-diploid (35–57 chromosomes) or near-tetraploid (81–103 chromosomes). The DNA index (DNI) of a tumor is the ratio of the number of chromosomes present to a normal diploid number of chromosomes (i.e., 46). Therefore, diploid cells have a DNA index of 1.0, whereas near-triploid cells have a DNA index ranging from 1.26 to 1.76. Near-triploid or hyperdiploid tumors are characterized by almost three complete haploid sets of chromosomes with few structural abnormalities. Importantly, patients with near-triploid tumors typically have favorable clinical and biologic prognostic factors and excellent survival rates, as compared with those patients who have near-diploid or near-tetraploid tumors. ^, This association is most important for infants with advanced disease as the prognostic significance of tumor ploidy appears to be lost in patients older than 2 years. Currently, ploidy/DNI affects the risk-stratification of limited groups of patients with stage L2, M, or MS neuroblastoma.

Segmental chromosomal alterations (SCA) are copy number gains or losses of individual chromosomal segments or arms and have prognostic significance in neuroblastoma, particularly in patients without MYCN amplification. For neuroblastoma, segmental chromosomal alterations are currently defined as chromosome 1p deletion and/or 11q deletion and/or chromosome 17q gain. An INRG study demonstrated that inclusion of this segmental genomic profile in the risk-stratification of neuroblastoma improved prognostic stratification compared to individual chromosomal/genetic markers. Segmental chromosomal alterations are utilized in the risk-stratification of neuroblastoma for limited groups of patients with stage L2, M, or MS neuroblastoma.

Telomeres are protective caps of repetitive DNA that are added to the ends of chromosomes to prevent cellular senescence with DNA replication. Activation of telomere maintenance mechanisms, either by telomerase activation or alternative lengthening of telomeres (ALT) is associated with poor outcomes in neuroblastoma. The gene TERT codes for the protein component of the telomerase holoenzyme ribonucleoprotein complex, and transcription of TERT is the rate-limiting step in telomerase assembly and activation. Recurrent activating genomic rearrangements leading to TERT promoter activation and therefore TERT transcription are characteristic of high-risk neuroblastoma and associated with poor outcomes, even among high-risk patients. ^, Additionally, N-MYC binds to the TERT promoter to activate TERT transcription. Somatic pathogenic variants or multiexon deletions of the gene ATRX lead to ALT, which is a telomere maintenance mechanism found in high-risk neuroblastoma among older patients. Interestingly, ATRX alterations and MYCN amplification were found to be mutually exclusive molecular events due to cellular synthetic lethality, pointing to a unique biology driving the development of high-risk neuroblastoma in older patients. Telomere-maintenance mechanisms, although not currently part of risk-stratification, may be an appropriate biomarker for risk-stratification of neuroblastoma patients in the future.

Recent advances in the understanding of neuroblastoma molecular biology have improved the understanding of therapeutic resistance and response to treatment. For example, therapeutic resistance has been associated with epigenetic cellular plasticity, or reversible transitions between a more differentiated noradrenergic phenotype and a more primitive mesenchymal phenotype in response to treatment. Relapsed neuroblastomas were found to exhibit frequent pathogenic variants in members of the RAS-MAPK pathway. Serial profiling of circulating tumor DNA (aka liquid biopsy) in patients with high-risk neuroblastoma may enable identification of resistant clones with actionable genomic alterations including those in ALK or the RAS-MAPK pathway.

Pretreatment Risk Stratification and Therapy

After the INRG stage (L1, L2, M, or MS) for a patient is determined, one then proceeds to determine the pretreatment risk group that guides therapy. In 2021, the COG published a revised neuroblastoma risk classification system (version 2.0) derived from INRGSS, patient age, INPC histology, and a variety of centrally assessed tumor biomarkers ( MYCN amplification status, ploidy/DNI, segmental chromosomal alterations) in almost 5000 COG patients enrolled on the ANBL00B1 biology study and treated with modern-era therapy (Figs. 63.6 and 63.7 ). A statistical framework was utilized to determine the influence of these features on EFS and OS and to broadly stratify the groups into low, intermediate, and high risk of disease relapse and death.

A flowchart shows Children's Oncology Group neuroblastoma risk stratification version two based on I N R G S S stage, age, M YC N status, biology, and surgical outcomes. The flowchart shows the Children's Oncology Group neuroblastoma risk stratification version two point zero, organized by I N R G S S stages L 1 and L 2. For L 1, patients are stratified by age and tumor size. Infants under twelve months with tumors less than five centimeters may qualify for observation and are labeled provisional low risk. Those with tumors five centimeters or larger are assessed based on surgical outcomes. Gross total resection leads to low risk, while incomplete resection requires M Y C N status to determine risk. M Y C N negative cases remain low risk, while M Y C N positive are high risk. Patients twelve months or older follow a similar structure, where incomplete resection with M Y C N positivity results in high risk. Stage L 2 includes M YC N positive patients classified as high risk regardless of other features. M Y C N negative patients under eighteen months are further assessed based on tumor biology. Favorable biology leads to intermediate risk, while unfavorable biology retains intermediate classification. For those aged eighteen months to under five years, I N P C classification dictates risk, favorable histology leads to intermediate risk and unfavorable histology to high risk. For patients aged five years or older, differentiating tumors fall under intermediate risk, while undifferentiated or poorly differentiated tumors are high risk. — Fig. 63.6

A flowchart shows Children's Oncology Group neuroblastoma risk stratification for M and M S stages based on age, M Y C N status, symptoms, and tumor biology. The flowchart shows neuroblastoma risk classification for M and M S stages according to the Children's Oncology Group neuroblastoma risk stratification system. For M stage, children under twelve months are categorized as high risk if M Y C N is positive and intermediate risk if negative. Children aged twelve to under eighteen months with M Y C N positivity are also high risk. If M Y C N is negative, tumor biology determines risk; any unfavorable biology, such as undifferentiated histology, diploid index of one, or segmental chromosomal aberration results in high risk, otherwise, patients are classified as intermediate risk. Children aged eighteen months or older are classified as high risk regardless of M Y C N status. For M S stage, infants under twelve months with systemic M S related symptoms, including respiratory, hepatic, renal, or coagulopathic manifestations, are classified as intermediate risk. If asymptomatic, M Y C N positive patients are high risk. M Y C N negative patients are classified based on tumor biology, those with any unfavorable features are intermediate risk, while those with all favorable features are low risk. For children aged twelve to under eighteen months, M Y C N positivity again leads to high risk. M Y C N negative children are evaluated by biology, any unfavorable findings indicate high risk, while favorable features result in intermediate risk. — Fig. 63.7

Low-Risk Neuroblastoma

Patients with low-risk neuroblastoma have a 5-year EFS and OS of 90.7% and 97.9%, respectively, according to the most recent COG risk-stratification system. In general, low-risk neuroblastomas are L1 tumors (localized and without IDRFs) in patients less than 12 months of age that meet criteria for observation or L1 tumors in patients of any age that are completely resected. Although rare in L1 neuroblastomas, if MYCN amplification is detected this only results in high-risk disease if the tumors have not been completely resected. If an L1 neuroblastoma is incompletely resected and not MYCN -amplified, this will also result in low risk-stratification. L1 neuroblastomas are generally not biopsied at diagnosis and therefore most patients who will be risk-stratified to low-risk neuroblastoma and who are not eligible for observation alone will undergo upfront surgical resection ( Fig. 63.6 ). Low-risk neuroblastoma is treated by either observation or complete surgical resection without chemotherapy. L1 or L2 ganglioneuroma or ganglioneuroblastoma-intermixed tumors are classified as low-risk disease. In contrast, ganglioneuroblastoma-nodular tumors are risk-stratified like neuroblastomas, with genetic analyses being performed specifically on the nodular component. Most localized neuroblastomas have favorable biologic features and are successfully treated with excision alone. In addition, studies suggest that a subset of localized tumors will spontaneously regress, or remain dormant, and that these patients can be observed without any treatment. ^, Local recurrences, while they rarely occur, can generally be managed surgically. ^,

Small, localized neuroblastomas in young infants tend to regress spontaneously. Based on this observation, the COG study, ANBL00P2, included an arm of expectant observation for patients with these lesions to further define their natural history. This study was designed to test the hypothesis that close biochemical and sonographic observation could be safely applied in infants with small adrenal masses. Resection was reserved for those rare cases in which there was evidence of continued growth. To be eligible, infants with an adrenal mass had to be <6 months of age when the mass was first identified; the mass must have been <16 mL in volume, if solid, or <65 mL if at least 25% cystic; and disease must have been limited to the adrenal gland. The results from this study confirmed that expectant observation of infants with small adrenal masses leads to excellent 3-year EFS (97.7 ± 2.3%) and 100% OS while avoiding operative intervention in >80% of patients.

Localized tumors without any image-defined risk factors (L1) in patients younger than 1 year of age at the time of presentation and with a tumor <5 cm by imaging studies were eligible for observation-only in the COG study ANBL1232. This study expanded on the ANBL00P2 inclusion criteria by increasing the age cut-off from 6 to 12 months, increasing the tumor size from 3.1 to 5 cm, and allowing nonadrenal primaries to be observed. Patients may have nonadrenal tumors confirmed by either an MIBG scan or elevated levels of catecholamine metabolites and may be observed up to 96 weeks without a biopsy. If these patients show disease progression (>50% growth of tumor, doubling of urinary catecholamines), surgical resection once off protocol is recommended. Other low-risk neuroblastoma patients who are not eligible for observation or who decline enrollment should have their tumor resected. If the tumor is resected completely, as should be possible with most L1 tumors, no adjuvant therapy would be given, regardless of the tumor biologic factors.

Intermediate-Risk Neuroblastoma

Patients with intermediate-risk neuroblastoma have a 5-year event free and OS of 85.1% and 95.8%, respectively, according to the most recent COG risk-stratification system. Patients with intermediate-risk neuroblastomas are those that do not meet the criteria for low-risk or high-risk disease. Specifically, intermediate-risk neuroblastomas are comprised of several groups of patients according to INRG stage, age, and biologic features. Patients with INRG stage L1 tumors are never assigned intermediate risk according to the COG. Patients with L2 tumors (localized, but with IDRFs) that do not meet criteria for high-risk disease based on absence of MYCN amplification or (depending on patient age) other adverse biologic features are classified as intermediate risk. In addition, patients with stage M disease who are younger than 12 months old and have tumors without MYCN amplification are categorized as intermediate risk as are patients with stage M disease who are between 12 and 18 months old without MYCN amplification and without any adverse biology (Figs. 63.6 and 63.7).

The COG ANBL0531 study aimed to reduce therapy for groups of intermediate-risk neuroblastoma patients using a biology- and treatment response-based algorithm while maintaining excellent 3-year OS. Overall, patients with intermediate-risk neuroblastoma receive between 2 and 8 cycles of chemotherapy depending on biology and treatment-response based characteristics. Outcomes are extremely favorable in patients with intermediate-risk neuroblastoma, provided that a partial response (50%–90% tumor volumetric reduction), very good partial response (>90% tumor volumetric reduction), or complete response (no evidence of disease) is achieved during therapy. ^, This minimum threshold of a partial response can be achieved by chemotherapy and/or surgery. Whether outcomes differ if a partial response is achieved in response to only chemotherapy versus only surgery or the combination remains a potential question for future investigation.

The COG ANBL1232 protocol opened in 2014 with the aim of utilizing response and biology-based disease features to guide therapy for patients with non–high-risk neuroblastoma. This protocol guides the contemporary management of most patients with intermediate-risk neuroblastoma, who receive between 2 and 8 cycles of systemic chemotherapy and/or surgical resection. In this protocol, patients younger than 18 months who are asymptomatic and have tumors with favorable biology determined by biopsy are observed. If patients are symptomatic, age is considered as the next criteria: patients younger than 3 months receive immediate chemotherapy (with full staging within 1 month) with plans to perform the tumor biopsy when they are stable, whereas patients 3–18 months old undergo a tumor biopsy and proceed through a response-based algorithm to determine the length of treatment. ANBL1232 is also prospectively studying an objective scoring system in which values will be assigned to symptoms and laboratory results to generate a clinical score. The trial will evaluate gastrointestinal symptoms, respiratory compromise, venous return, renal compromise, and hepatic dysfunction. This protocol also guides the management of children with INRG stage MS disease. The rare infant with MS disease and either unfavorable Shimada histology or a DNA index of 1 (or if the biology is not known) are treated as having intermediate-risk disease. Those patients with MS disease that is MYCN -amplified are treated as having high-risk disease.

High-Risk Neuroblastoma

Patients with high-risk neuroblastoma have a 5-year EFS and OS of 51.2% and 62.5%, respectively, according to the most recent COG risk-stratification system. Most high-risk neuroblastomas are metastatic tumors in patients older than 18 months or tumors in any age patient with MYCN amplification (unless L1, MYCN -amplified, and completely resected). For patients with stage M tumors between 12 and 18 months of age with MYCN nonamplified tumors, any unfavorable biology (unfavorable histology, diploidy, or segmental chromosomal alterations) results in high-risk disease ( Fig. 63.7 ).

In contrast to low- and intermediate-risk neuroblastoma, patients with high-risk neuroblastoma are treated with intense multimodality systemic and local control therapies including multiagent induction chemotherapy, surgery, radiation therapy, myeloablative chemotherapy with stem cell rescue, immunotherapy with an anti-GD2 antibody, and maintenance therapy with a retinoic acid-based differentiating agent ( Table 63.4 ). The therapy for high-risk neuroblastoma involves three phases: induction including local control, consolidation, and maintenance with treatment of minimal residual disease using biologic agents. Stem cell harvest is typically performed after the first two cycles of induction therapy, and resection of the primary tumor and locoregional disease is attempted after the fourth or fifth cycle of chemotherapy. At diagnosis, neuroblastoma is generally a chemotherapy-sensitive tumor, and multiagent chemotherapy is usually effective in achieving at least a partial response in children with disseminated disease. However, despite this approach, nearly half of high-risk neuroblastoma patients ultimately die from their disease. More recent and ongoing high-risk neuroblastoma COG clinical trials have aimed to augment the ability to achieve an end-induction partial response by incorporating a variety of agents into the induction phase of therapy including anti-GD2 antibody therapy, MIBG therapy, and ALK -targeted therapies for tumors with ALK alterations.

Table 63.4

Representative Components of Intensive Multimodality Therapy for High-Risk Neuroblastoma

Phases of Therapy	Therapeutic Modalities	Notes
1. Induction Therapy with Local Control	Multiagent induction chemotherapy	Agents typically include: cisplatin, cyclophosphamide, doxorubicin, etoposide, topotecan, vincristine
	Investigational agents	Investigational agents used on recent Children’s Oncology Group Clinical Trials: anti-GD2 antibody during induction, ¹³¹ I-MIBG therapy, ALK-targeted therapies (e.g., crizotinib)
	Stem cell collection
	Surgical resection	Typically performed after 4th–5th cycle of chemotherapy
	Primary site radiotherapy
2. Consolidation Therapy	Myeloablative chemotherapy with autologous tandem stem cell transplant	Myeloablative regimens may include: cisplatin/etoposide/melphalan, busulfan/melphalan, or thiotepa/cyclophosphamide plus cisplatin/etoposide/melphalan
3. Postconsolidation Therapy	Anti-GD2 antibody therapy	Concomitant IL-2 is omitted in contemporary regimens
	13- cis -retinoic acid

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