Case of the Month: Paravertebral

This case first appeared as Performance Improvement Program in Surgical Pathology (PIP) 2015, case 02, and is a ganglioneuroblastoma.

The histologic sections reveal a prominent proliferation of spindle-shaped cells (fibroblasts) variably associated with organized fascicles of neuritic processes and mature Schwann cells (consistent with Schwannian stroma). This mature stroma is intermixed with aggregates of immature and maturing neuroblasts (including mature ganglion cells); a neuropil background is noted in some areas. The Schwannian stroma comprises more than 50% of the tumor and the immature elements comprise close to 10% of the tumor. Also noted are small areas of hemorrhage and a few stromal aggregates of lymphocytes. The microscopic findings are consistent with those of a ganglioneuroblastoma, intermixed (Schwannian stroma-rich). Neuroblastic tumors: The neuroblastic tumors (neuroblastoma [NBL], ganglioneuroblastoma [GNB] and ganglioneuroma [GN]) are a group of neoplasms derived from primordial neural crest cells of the sympathetic nervous system. These tumors have different histological characteristics. However, they share some unique features, including spontaneous or therapy induced maturation and/or spontaneous tumor regression.

NBL is the most common extracranial solid tumor of childhood and the most frequently diagnosed tumor of infancy; the median age at the time of diagnosis is 18 months. NBLs are slightly more common in males and less common in African-Americans. Approximately 650 new cases of NBL are diagnosed in the USA every year. The clinical presentation is variable, ranging from small asymptomatic lesions to large masses associated with widely disseminated disease. Close to 90% of NBLs produce catecholamines and the metabolites can be detected in the patient's serum and urine. In some cases, NBLs can be seen in association with other disorders, including Turner syndrome, neurofibromatosis, and Hirschsprung disease. In the majority of cases NBLs present in the adrenal medulla (35%). Other sites of primary involvement include the extra-adrenal retroperitoneum (30%–35%), the posterior mediastinum (20%) and the paravertebral region (3%–5%).

Although most NBLs occur sporadically, between 1% and 2% of cases are familial or hereditary. In this setting the neoplasms may involve multiple primary autonomic sites, including both adrenal glands. Germ-line mutations in the ALK (anaplastic lymphoma kinase) oncogene have been identified in most cases of familial NBLs as well as in a smaller subset of sporadic tumors. Loss of function mutations in the homeobox gene PHOX2B were described in children with either sporadic or familial NBL associated with congenital central hypoventilation syndrome, Hirschsprung disease, or both. Other discoveries associated with familial NBL include a hereditary NBL predisposition gene (HNB1) in the distal short arm of chromosome 16p (16p12-13), and another gene on the distal short arm of chromosome 4p (4p16).

Following a modification of the original 1984 Shimada Classification by the International Neuroblastoma Pathology Committee (INPC) in 1999, peripheral neuroblastic tumors were classified into four categories:

• Neuroblastoma (Schwannian stroma-poor): Grossly, these tumors can be well circumscribed single nodules or multinodular masses. They vary in size from minute tumors to discrete lesions up to 10 cm in greatest dimension. The cut surfaces are usually soft, tan-yellow or tan gray, and may exhibit areas of hemorrhage, necrosis, calcification, and/or cystic change. Microscopically they are composed of small round to oval neoplastic cells with scanty cytoplasm, round to oval nuclei with “salt and pepper” chromatin, and poorly defined cell borders. The neoplastic cells are not uncommonly arranged in a lobular pattern associated with fine fibrovascular tissue septae, but may also be arranged in solid sheets. Brisk mitotic activity, karyorrhexis and cellular pleomorphism may be prominent features of some tumors. The background of the cellular process is variably composed of neuropil, an eosinophilic fibrillary network corresponding to the neuritic processes of the neuroblasts. Homer-Wright rosettes may be seen, characterized by neoplastic cells arranged around a central neuropil-filled space. Also present in some tumors are neuroblasts differentiating towards ganglion cells; these more mature cells exhibit large eccentrically located vesicular nuclei with prominent nucleoli associated with more abundant eosinophilic/amphophilic cytoplasm and less prominent neuritic processes (neuropil). NBLs are defined as being "Schwannian stroma-poor". Schwannian stroma refers to a proliferation of fibroblasts associated with organized fascicles of neuritic processes and mature Schwan cells.

When microscopic foci of neuroblasts are noted in or around the adrenal glands they are referred to as “in situ” NBLs. Because most of these lesions are asymptomatic incidental findings (and a large number of them spontaneously regress), some investigators question their true nature (neoplastic vs. premalignant vs. developmental anomaly/rest). NBLs are further subclassified into "undifferentiated", "poorly differentiated", and "differentiating" subtypes. The "undifferentiated" subtype is characterized by undifferentiated neuroblasts; neuropil and ganglionic differentiation are less prominent than in the other subtypes. A diffuse growth pattern is commonly seen. The "poorly differentiated" subtype is the most common pattern. Differentiating neuroblasts comprise <5% of these tumors; neuropil and Homer-Wright rosettes are common findings. A lobular growth pattern is commonly seen. In the "differentiating subtype" >5% of the tumor cells are differentiating neuroblasts, and neuropil is a prominent feature. Rare variants of NBLs with aggressive clinical behavior and poor, often fatal, outcomes also exist. One of these variants exhibits anaplastic cellular changes and another is composed of large cells with prominent nucleoli.

• Ganglioneuroblastoma, intermixed (Schwannian stroma-rich): Grossly, these tumors are usually nodular and firm, with a tan-yellow homogeneous cut surface that may exhibit fibrous bands. Microscopically, they are defined as being "Schwannian stroma-rich". They are composed of foci of neuroblasts displaying varying stages of differentiation and associated with neuropil. The Schwannian stroma should occupy >50% of the tumor. It is not uncommon to identify mature ganglion cells associated with the Schwannian stroma in a pattern identical to that noted in ganglioneuromas (see below). Also noted occasionally are small stromal aggregates of mature lymphocytes.
• Ganglioneuroma (Schwannian stroma-dominant): Grossly, these tumors exhibit features similar to those described in cases of GNB intermixed (Schwannian stroma-rich). Microscopically, they are defined as being "Schwannian stroma-dominant". They are composed of a minor component of maturing/mature ganglion cells, arranged in clusters or as single cells, associated with a prominent Schwannian stroma. The GNs are further subclassified into "maturing" and "mature" subtypes. In the "maturing" subtype (previously classified as "ganglioneuroblastoma, well differentiated" in the Shimada Classification) the minor component consists of mature ganglion cells and dispersed individual immature ganglion cells and/or neuroblasts. In the "mature" subtype the minor component consists of mature ganglion cells surrounded by satellite cells. Also noted occasionally are small stromal aggregates of mature lymphocytes.
• Ganglioneuroblastoma, nodular (Schwannian stroma-dominant/stroma-rich and stroma poor): This is considered a "composite" tumor. In this setting one or more neuroblastic nodules co-exist with GNB-intermixed or with GN. The nodular quality of these tumors can be evaluated grossly, with the more immature/malignant nodules exhibiting areas of hemorrhage and/or necrosis alternating with homogeneous tan-yellow nodules of mature/better differentiated elements. However, sometimes the gross features are not that distinct, requiring extensive tumor sampling in order to further characterize the components of the tumor following microscopic evaluation.

Evaluation of neuroblastic tumors: Part of the histological evaluation of a NBL includes the determination of the mitotic-karyorrhexis index (MKI) of the tumor, as recommended by the INPC. The MKI is determined by counting the number of neoplastic cells undergoing mitosis and those in the process of karyorrhexis and is defined as low (<2% or <100 mitotic and karyorrhectic cells per 5,000 neuroblasts), intermediate (2% to 4%, or 100 to 200 mitotic and karyorrhectic cells per 5,000 neuroblasts), and high (>4% or >200 mitotic and karyorrhectic cells per 5,000 neuroblasts). Increased MKI correlates with MYCN amplification.

The INPC also developed the International Neuroblastoma Pathology Classification, later revised in 2003. This classification distinguishes two pathologic-prognostic groups of patients: one with favorable histology and another with unfavorable histology. This classification is age-linked and uses three histological indicators: status of Schwannian stromal development, grade of neuroblastic differentiation, and MKI. The evaluation should be performed on a resected specimen or a biopsy prior to any treatment (chemotherapy and/or radiation). Metastatic specimens are also eligible for evaluation prior to therapy, except for bone marrow biopsies (not considered adequate for MIK determination).

* The revised INCP classification (2003) distinguishes a favorable and unfavorable subset by applying the same age-linked evaluation to the nodular (NBL) components of the tumors in this category.

In cases of NBLs, several factors affect patient outcome and are associated with favorable or unfavorable prognosis. These prognostic (or risk) factors include age, stage, MYCN status, histology, and DNA ploidy. It is based on these "core" prognostic factors that patients are classified as being either "low", "intermediate", or "high" risk.

• Age: Patients younger than 18 months have an excellent prognosis independent of the clinical stage and are considered “low” risk (favorable prognosis). Children older than 18 months have an unfavorable prognosis and are considered "intermediate" risk; if they have additional unfavorable risk factors (like MYCN amplification) they are considered "high" risk.
• Stage: The International Neuroblastoma Staging System (INSS) is a widely used postsurgical staging system used for prognostic purposes. When this system is used, clinical stages 1, 2A, 2B, and 4S are considered to have a favorable prognosis irrespective of age, except when the tumors exhibit MYCN amplification (see below). Patients determined to be stages 3 or 4 have an unfavorable prognosis.
• MYCN amplification: Amplification of the MYCN oncogene has a very important impact in prognosis. When MYCN amplification is present, the tumor is elevated to a "high" risk category, irrespective of the patient's age, his/her clinical stage, or the histological features of the tumor. MYCN amplification is noted in 20% to 30% of primary tumors; the degree of amplification correlates with a worse prognosis. The MYCN amplification status usually remains unchanged following chemotherapy treatment.
• Histology: This independent prognostic factor highlights the importance of accurate histological evaluation when examining neuroblastic tumors. The presence of Schwannian stroma and ganglionic differentiation is considered a favorable prognostic indicator, but its absence is considered unfavorable. Because neuroblastic tumors may demonstrate variable histological features, extensive sampling is recommended (1 tumor section per centimeter of the largest dimension of the tumor). Immunohistochemical stains can also assist in confirming the nature of a neuroblastic tumor. Neuroblasts are immunopositive for several markers, including neuron-specific enolase, NB84, PGP9.5, synaptophysin, chromogranin, CD57, S-100 protein, and tyrosine hydroxylase; the Schwannian stroma is immunopositive for S-100 protein. These immunostains may be part of a panel when the differential diagnosis includes one of these lesions.
• DNA ploidy: This independent prognostic factor correlates with outcome only for children under the age of 2, as it loses its prognostic significance in children above that age. Tumors with hyper-diploid or near triploid DNA contents are considered lesions with a favorable prognosis. Tumors with near diploid DNA content are considered lesions with an unfavorable prognosis. Other unfavorable prognostic factors include the presence of chromosome 17q gain, chromosome 1p loss, chromosome 11q loss, and TrkB (tropomyosin-related kinase B) expression as well as the absence of TrkA (tropomyosin-related kinase A) expression.

Differential diagnosis and treatment: NBLs, especially the undifferentiated type, are in the differential diagnosis of the "small blue round cell tumors of childhood". These lesions include lymphoblastic lymphoma/leukemia, Wilms tumor, alveolar rhabdomyosarcoma, desmoplastic small round cell tumor, and Ewing sarcoma/primitive neuroectodermal tumor. Immunohistochemical stains (i.e., myogenin, MyoD1, CD99, lymphoid markers, desmin) together with detailed histological features (identified in properly sampled and well preserved tumors) and the clinical presentation are likely to assist in differentiating these neoplasms from NBLs.

Several methods can be used when treating NBLs and GNBs, including surgery, chemotherapy, radiotherapy, and observation. However, before choosing any therapeutic modality, clinicians consider numerous factors in order to assign a patient to one of the three risk groups described above. This practice allows the use of treatment options designed to increase the chances of curing the patient and preventing tumor recurrence, while minimizing side effects.

1. In cases of neuroblastomas, several factors affect patient outcome and are associated with favorable or unfavorable prognosis. Which of the following do these "core" prognostic (or risk) factors include?
   
   1. Age, race, sex, and urine cathecholamine levels
   2. Age, stage, MYCN amplification status, histology, and DNA ploidy
   3. An association with Turner syndrome, neurofibromatosis, or Hirschsprung disease
   4. Histology, germline mutations in the ALK (anaplastic lymphoma kinase) gene, and low or absent telomerase expression
   5. Immunopositivity with S-100 protein and tyrosine hydroxylase, and the presence of Schwannian stroma
2. The International Neuroblastoma Pathology Classification distinguishes two pathologic-prognostic groups of patients: one with favorable histology and another with unfavorable histology. This classification is age-linked and uses which three of the following histological indicators?
   
   1. Absent Schwannian stromal development, formation of Homer-Wright rosettes, and low mitotic-karyorrhexis index
   2. Necrosis, hemorrhage and ganglionic differentiation
   3. Positive immunostaining for MyoD1, grade of neuroblastic differentiation, and high mitotic-karyorrhexis index
   4. Positive immunostaining for PGP9.5, tumor necrosis, and MYCN amplification
   5. Status of Schwannian stromal development, grade of neuroblastic differentiation, and mitotic-karyorrhexis index
3. The mitotic-karyorrhexis index (MKI) of a neuroblastoma is determined by counting the number of neoplastic cells undergoing mitosis and those in the process of karyorrhexis and is defined as low, intermediate and high. An intermediate MKI is reported when which of the following is determined?
   
   1. <2% or < 100 mitotic and karyorrhectic cells per 5,000 neuroblasts
   2. 2% to 4%, or 100 to 200 mitotic and karyorrhectic cells per 5,000 neuroblasts
   3. 2% to 10%, or 100 to 400 mitotic and karyorrhectic cells per 5,000 neuroblasts
   4. >4% or >200 mitotic and karyorrhectic cells per 5,000 neuroblasts
   5. 6% to 10%, or 300 to 600 mitotic and karyorrhectic cells per 5,000 neuroblasts

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2015
Nilsa C. Ramirez, MD
Surgical Pathology Committee
Nationwide Children's Hospital
Columbus, OH 

1. Age, stage, MYCN amplification status, histology, and DNA ploidy (b). 
2. Status of Schwannian stromal development, grade of neuroblastic differentiation, and mitotic-karyorrhexis index (e). 
3. 2% to 4%, or 100 to 200 mitotic and karyorrhectic cells per 5,000 neuroblasts (b).