Neuroblastoma Workup

General laboratory studies should be routinely obtained in children suspected of having neuroblastoma. Results are as follows:

• A complete blood cell count (CBC) should be obtained to determine if the child has anemia, which typically does not occur until the tumor has become widely disseminated; in patients with overwhelming bone marrow involvement, thrombocytopenia may also be present

• Once dissemination occurs, abnormalities in findings of coagulation studies (prothrombin time [PT], activated partial thromboplastin time [aPTT]) may occur secondary to liver involvement

• The erythrocyte sedimentation rate, a nonspecific acute-phase reactant, is elevated in classic neuroblastoma

Elevated metabolic catecholamine by-products can be detected in the urine of patients with neuroblastoma. The presence of these by-products serves as useful inclusion criteria when the diagnosis of neuroblastoma is being considered.

Phenylalanine and tyrosine are catecholamine precursors, which are converted through a sequence of enzymatic events to dihydroxyphenylalanine (DOPA), dopamine, norepinephrine, and epinephrine. DOPA and dopamine are metabolized into their final product, homovanillic acid (HVA), while norepinephrine and epinephrine are metabolized into vanillylmandelic acid (VMA).

Ninety percent of neuroblastoma tumors secrete these by-products. This fact becomes clinically relevant because children with dedifferentiated tumors excrete higher levels of HVA than VMA. This occurs because dedifferentiated tumors have lost the final enzymatic pathway that converts HVA to VMA. A low VMA-to-HVA ratio is consistent with a poorly differentiated tumor and indicative of a poor prognosis.

Neuroblastoma cells lack the enzyme that converts norepinephrine to epinephrine. Despite this fact, elevated levels of norepinephrine are not identified in the serum of patients with neuroblastoma. This might be explained by at least two processes: (1) norepinephrine may be catabolized within the tumor; and/or (2) tyrosine hydrolase, the initial enzyme in catecholamine synthesis, is subject to a negative feedback loop by norepinephrine.

A LaBrosse VMA spot test may be used to screen patients in certain institutions. It is economical but has low sensitivity and specificity.

High-performance liquid chromatography has a much lower false-positive rate and is more sensitive than the LaBrosse VMA spot test, but it is more expensive and is therefore often used only to confirm a positive result on a spot test.

Nonspecific tumor markers can be identified in patients with neuroblastoma. ^[18] Neuron-specific enolase (NSE), lactic dehydrogenase (LDH), and ferritin are markers useful in the identification of active disease, as well as in prognostication. Approximately 96% of patients with metastatic neuroblastomas demonstrate an elevated NSE level, which has been associated with a poor prognosis.

Radiographic assessment is recommended in all infants and children with an abdominal mass. The standard diagnostic imaging modalities include the following:

• Plain abdominal radiography (kidneys, ureters, bladder [KUB])

• Renal/bladder ultrasonography

• Bone scintigraphy

• Computed tomography (CT) or magnetic resonance imaging (MRI)

KUB most commonly reveals finely stippled calcifications of the abdomen or posterior mediastinum.

Renal/bladder ultrasonography improves the diagnostic evaluation and is probably the single best imaging modality to obtain. Ultrasonography is noninvasive and provides relevant information regarding the laterality, consistency, and size of the mass.

Abdominal CT scanning or MRI usually follows ultrasonography. Both of these studies are more invasive, in that they require general sedation for young children. The benefit is that they enhance the ultrasonographic findings by providing information about regional lymph nodes, vessel invasion, and distant metastatic disease. See the images below.

CT scan in a 2-week-old boy noted to have an abdominal mass on a prenatal sonogram. This postnatal abdominal CT scan revealed a left suprarenal mass with mass effect of the spleen.

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Abdominal CT scan in a 2-week-old boy noted to have an abdominal mass on a prenatal sonogram. A postnatal abdominal CT scan revealed a left suprarenal mass with mass effect of the spleen (see the previous image). This abdominal CT scan represents a more caudal view. Note the very large left mass with central necrosis. The mass effect of the spleen is apparent.

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A 2-week-old boy is noted to have an abdominal mass on prenatal ultrasound. A postnatal abdominal CT scan revealed a left suprarenal mass with mass effect of the spleen (see the first image above). A more caudal view revealed the very large left mass with central necrosis (see the second image above). This is a more caudal view of the CT scan than in the previous 2 images. The left kidney comes into view, as it is inferiorly displaced and laterally rotated by the large superior neuroblastoma.

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Bulky lymph nodes just medial to the left kidney.

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Bone scintigraphy and a skeletal survey to detect cortical bone disease are helpful in the diagnosis of neuroblastoma. Metaiodobenzylguanidine (MIBG) is a compound taken up by catecholaminergic cells that competes for uptake even in neuroblastoma cells. In this way, MIBG is quite sensitive and specific in the detection of metastasis to bones and soft tissue, with highest sensitivity (91-97%) in the detection of bone deposits.

Bone scintigraphy using 99Tc diphosphonate and a skeletal bone survey to detect cortical bone disease are essential if MIBG scintigraphy results are negative in the bone. MIBG is recommended for re-assessment both during and after therapy in high-risk patients with MIBG-avid disease at diagnosis.

Expression of somatostatin (SS) receptors has been described in neuroblastoma cell lines and tumors. Studies have shown that these tumors can be successfully targeted with radioactive SS analogs as a method of detection. Currently, the indication for scanning with radio-labeled SS analog in children with neuroblastoma is not well-defined because this method is less sensitive than MIBG scanning (64% vs 94%). However, because the presence of neuroblastoma SS receptors is associated with favorable clinical and biological prognostic factors, radio-labeled SS analog could provide valuable information. In fact, improved survival has been found in patients with SS receptor–positive neuroblastoma. However, more studies need to be performed to confirm the benefits of SS receptor scanning.

Biopsy is the sine qua non in the diagnostic evaluation of neuroblastoma. To confirm the diagnosis of neuroblastoma, histologic evidence of neural origin or differentiation is required. Samples of tumor tissue can be viewed via light or electron microscopy or via immunohistochemistry. Although open surgical biopsy has traditionally been advocated, Mullassery and colleagues reported that image-guided needle biopsy can also yield adequate tissue samples. ^[19]

Another option is to sample bone marrow, a frequent metastatic site for neuroblastoma. The literature is confusing in terms of the number of bone marrow aspirates or biopsies needed to diagnose neuroblastoma. An international committee on neuroblastoma staging has recommended obtaining two bone marrow aspirates and two biopsies, one from each posterior iliac crest.

The issue of biopsies might become obsolete because immunocytology of marrow aspirates may offer the single best source of diagnostic information. Recently, a large body of published work has addressed the use of immunocytochemical and polymerase chain reaction (PCR)–based technologies to detect neuroblastoma cells and neuroblastoma-specific transcripts such as tyrosine hydroxylase and disialoganglioside (GD2) synthase in marrow or blood samples. This method is used to assess minimal disease during the course of treatment. Although these techniques can greatly increase sensitivity, whether this increased sensitivity provides prognostic information about the likelihood of relapse is still unclear.

The three distinct histologic patterns of the neurocristopathies include neuroblastoma, ganglioneuroblastoma, and ganglioneuroma. They represent a spectrum of maturation and dedifferentiation. The typical neuroblastoma is characterized by small uniform cells that contain dense hyperchromatic nuclei and scant cytoplasm. A neuritic process called neuropil is pathognomonic of all except the most primitive neuroblastoma. Homer-Wright pseudorosettes are clusters of neuroblasts surrounding areas of eosinophilic neuropil and are observed in 15-50% of patients. If identified, they are diagnostic of neuroblastoma.

The minimal diagnostic criteria for diagnosing neuroblastoma have been established by an international group of conferees and corresponding participants. These criteria include (1) unequivocal pathologic diagnosis or (2) unequivocal bone marrow (syncytia) and elevated levels of urinary catecholamine metabolic by-products. Both of these diagnostic criteria require a histopathologic diagnosis.

Molecular pathogenesis

The cancer genes most commonly altered in adult carcinogenesis (eg, TP53, CDKN2A, ras) are rarely aberrant in neuroblastoma. TP53 -inactivating mutations are uncommon in primary tumors due to neuroblastoma, although they have been documented in cell lines among patients with relapsing neuroblastoma. Thus, with the exception of N-myc, major pathways of human neoplasia do not seem to be deregulated. Indeed, the only reliable genetically engineered murine model of neuroblastoma results from targeted overexpression of human N-myc cDNA to the murine neural crest.

Activating mutations in the tyrosine kinase domain of the anaplastic lymphoma kinase (ALK) gene occur in the majority of hereditary neuroblastoma cases, and also in some sporadic cases of advanced neuroblastoma. Subclonal ALK mutations can be present at diagnosis, with subsequent clonal expansion at relapse ^[20]

At least six different staging systems for neuroblastoma exist. Historically, each staging system represents a temporal improvement in the understanding of the tumor. However, the presence of so many systems has not only confounded the literature but also complicated the comparison of studies between institutions. Twenty years ago, the International Neuroblastoma Staging System (INSS) was established to provide a uniform staging system.

The INSS is a clinical, radiographic, and surgical appraisal of children with neuroblastoma. The system combines many of the most important diagnostic criteria from each of the staging systems and includes initial distribution and surgical resectability of the tumor.

Specific requirements to stage neuroblastoma include the following:

• Bone marrow aspirates and biopsy samples

• Body CT scan (excluding head, if not clinically indicated)

• Bone scan

• MIBG scintigraphy

Arabic numerals are used to distinguish INSS staging from other systems. INSS stages are as follows:

• Stage 1 is characterized by a localized tumor with complete gross excision, with or without microscopic residual disease; representative ipsilateral lymph nodes that test negative for tumor are present microscopically (nodes attached to and removed with the primary tumor may test positive)

• Stage 2A is characterized by a localized tumor with incomplete gross excision; representative ipsilateral nonadherent lymph nodes that test negative for tumor are present microscopically

• Stage 2B is characterized by a localized tumor with or without complete gross excision, with ipsilateral nonadherent lymph nodes that test positive for tumor; enlarged contralateral lymph nodes must test negative microscopically

• Stage 3 is an unresectable unilateral tumor infiltrating across the midline, with or without regional lymph node involvement; a localized unilateral tumor with contralateral regional lymph node involvement; or a midline tumor with bilateral extension by infiltration (unresectable) or by lymph node involvement (the midline is defined as the vertebral column; tumors that originate on one side and cross the midline must infiltrate to or beyond the opposite side of the vertebral column)

• Stage 4 is any primary tumor disseminated to distant lymph nodes, bone, bone marrow, liver, skin, and/or other organs (except as defined for stage 4S)

• Stage 4S (S = special) occurs in infants younger than 12 months and is characterized by a localized primary tumor (as defined for stage 1, 2A, or 2B), with dissemination limited to skin, liver, and/or bone marrow

Other features of stage 4S include the following:

• Marrow involvement should be minimal (ie, < 10% of total nucleated cells identified as malignant via bone biopsy or bone marrow aspirate); more extensive bone marrow involvement is considered stage 4 disease

• The results of the MIBG scan (if performed) should be negative for disease in the bone marrow.

Stage 4S is the most unusual group, comprising approximately 5% of patients with neuroblastoma. All else being equal, these children would normally be classified as having stage 1 or 2 disease; however, disease in this special group of infants almost always spontaneously regresses. Nonetheless, infants younger than 2 months frequently develop extensive and rapidly progressive intrahepatic expansion of neuroblastoma that can result in respiratory compromise. The 5-year survival rate in patients with stage 4S disease is 75%.

Stage for stage, infants with neuroblastoma have a better prognosis than older children. In fact, statistically, age is the most significant clinical prognosticator for neuroblastoma. Forty percent of infants (< 1 y) have localized neuroblastoma, compared with 20% of children older than 1 year. Additionally, nearly 70% of children older than 1 year have disseminated neuroblastoma, compared with less than 25% of infants.

The Children’s Oncology Group (COG) uses the major prognostic factors to place children into three risk groups: low, intermediate, and high. ^[21] These risk groups are used to guide treatment selection. Five-year survival rates by risk group are as follows:

• Low risk: > 95%

• Intermediate risk: Approximately 90-95%

• High risk: Approximately 40-50%

Low-risk group criteria are as follows:

• Stage 1 disease

• Stage 2A or 2B disease, patient age younger than 12 months

• Stage 2A or 2B disease, patient age older than 12 months, no extra copies of the MYCN gene

• Stage 4S disease, favorable histology, hyperdiploid, no extra copies of MYCN

Intermediate-risk group criteria are as follows:

• Stage 3 disease, patient age younger than 12 months; no extra copies of MYCN

• Stage 3 disease, patient age older than 12 months, no extra copies of MYCN, favorable histology

• Stage 4 disease, patient age younger than 12 months, no extra copies of MYCN

• Stage 4S disease, no extra copies of MYCN, normal DNA ploidy, and/or unfavorable histology

High-risk group criteria are as follows:

• Stage 2A or 2B disease, patient age older than 12 months, extra copies of MYCN

• Stage 3 disease, patient age younger than 12 months, extra copies of MYCN

• Stage 3 disease, patient age older than 12 months, extra copies of MYCN

• Stage 3 disease, patient age older than 18 months, unfavorable histology

• Stage 4 disease, extra copies of MYCN

• Stage 4 disease, patient age older than 18 months

• Stage 4 disease, patient age between 12 and 18 months, extra copies of MYCN, unfavorable histology, and/or normal DNA ploidy (DNA index of 1)

• Stage 4S disease, extra copies of MYCN