2020 NPB Minisymposium

This minisymposium was originally published in 2020. The information provided in this minisymposium was accurate and correct at the time of initial program release. Any changes in terminology since the time of initial publication may not be reflected in this minisymposium.

The phrase “small round blue cell” is ubiquitous in surgical pathology because it provides a succinct description of a category of look-alike entities. These lesions are composed of round cells with an increased nuclear-to-cytoplasmic (N:C) ratio, and their small round cell appearance often suggests a lack of differentiation (ie, embryonal or primitive histogenesis). In practice, the term is sometimes applied to tumors in which the cells are not exactly “small” or “round” but, nonetheless, demonstrate high cellularity and are tightly packed with high N:C ratios (Image 1). Small round blue cell tumors encountered in the CNS include malignancies primary to the CNS as well as hematological, mesenchymal, and metastatic tumors. The breadth of the differential diagnosis, despite a strikingly similar histologic appearance, makes this category particularly challenging for the surgical pathologist, especially in the intraoperative setting. The key to effective workup and diagnosis of these lesions is familiarity with both common and uncommon disorders sharing this microscopic appearance.

Ideally, the pathologist should be aware of the clinical scenario prior to evaluating a specimen. History of past malignancies or knowledge of concurrent systemic lesions may make an otherwise difficult differential a straightforward exercise. At the minimum, knowing the location of the lesion (supratentorial versus infratentorial, intraparenchymal/intra-axial, leptomeningeal/dural, or skull-based) can refine the differential diagnostic considerations significantly. Additionally, the age of the patient is key, as some of the entities described below are exceedingly rare outside of specific age ranges.

These lesions are usually first encountered at the time of intraoperative consultation or, occasionally, on analysis of CSF cytology. Small round blue cell tumors typically have a nonspecific cytologic appearance. Tumor cells are usually discohesive without cytoplasmic processes or a fibrillary background on smear or squash preparations. Apoptotic bodies and/or mitotic figures may be recognized since many small round cell tumors are highly proliferative. Frozen section may reveal only sheets of cells without a specific architectural pattern. Furthermore, these tumors are often prone to crush or smear artifact and are particularly sensitive to cautery effect. Because many different histologic entities can demonstrate similar features, definitive diagnosis at the time of intraoperative or cytologic interpretation is frequently not possible and should be discouraged and deferred. Fortunately, the term “small round blue cell tumor” is highly familiar to most neurosurgeons and provides a valuable piece of potentially actionable information. Therefore, it is preferable to use that term when appropriate instead of simply stating “lesional tissue identified.” For example, in some clinical scenarios, an intraoperative diagnosis of small round blue cell tumor may alert the surgeon to the possibility of lymphoma and limit aggressive pursuit of complete total resection. Please refer to Table 1 at the end for a summary of diagnostic entities to consider in the differential of a small round blue cell tumor, based on age and location.

Table 1: Differential diagnosis of small round blue cell tumors of the central nervous system.

Metastases

Metastatic malignancies are among the most frequently encountered diagnostic entities in the CNS of older adults. One such entity with small round blue cell morphology is metastatic small cell carcinoma. Fortunately, the diagnosis can be easily made with the appropriate use of keratin and neuroendocrine markers. Another neoplasm that can occasionally have a small cell appearance is melanoma. A diagnosis of metastatic melanoma can be supported by immunoreactivity for HMB45, MITF, or MART1. S100 positivity, while sensitive for melanomas overall, is not useful in the brain due to its expression in glial and neuronal cells and neoplasms.

Additional CNS metastases include other carcinomas, sarcomas, and involvement by systemic lymphomas. Detailed discussion of these neoplasms and their differential diagnoses is beyond the scope of this minisymposium. When working up a small round cell neoplasm that does not provide obvious diagnostic clues, it is important to keep a wide differential diagnosis and employ lineage markers that span various metastatic and primary lesions as a first line of workup.

Embryonal Tumors of the CNS

Among primary CNS tumors in the pediatric population, the most commonly encountered entity with a small round blue cell appearance is medulloblastoma. Medulloblastoma accounts for nearly 20% of pediatric brain tumors but can also occur in adults. The key histologic features of medulloblastoma include dense cellularity, small undifferentiated cells, mild-to-moderate pleomorphism, and a high mitotic count (Image 1A). According to the 2016 revision of the World Health Organization Classification of Tumours of the Central Nervous System, medulloblastomas should now be subcategorized by histologic appearance (classic, desmoplastic/nodular, with extensive nodularity, or large cell/anaplastic) as well as by molecular genetic features (WNT-activated, SHH-activated, or non-WNT/non-SHH), if possible. The genetic subgroups of medulloblastoma can be determined by immunophenotype and/or associated molecular and cytogenetic alterations.

There are no reliable histologic or cytologic features to differentiate medulloblastomas from atypical teratoid/rhabdoid tumors (AT/RTs), and both may occur in the posterior fossa of young children. For this reason, routine screening with IHC for INI1/SMARCB1 is advisable before rendering a new diagnosis of medulloblastoma. The loss of immunoreactivity for INI1/SMARCB1 reflects the key feature of AT/RT: inactivation of the SMARCB1 gene. Exceptionally, AT/RT can arise from inactivation of the related gene SMARCA4. Only about half of AT/RTs occur in the posterior fossa; any part of the neuraxis may be involved. While areas of rhabdoid cytology or other heterologous elements (epithelial or mesenchymal) are helpful features when present, not all AT/RTs contain them. A small round blue cell tumor in a patient under the age of 3 years should raise consideration for AT/RT. The median age at diagnosis for AT/RT is 1 to 2 years, with even earlier occurrences in

the setting of germline SMARCB1 mutation (rhabdoid tumor predisposition). Children with germline SMARCB1 mutations may have synchronous rhabdoid tumors of the kidney or other soft tissues. Other immunostains can be helpful in AT/RT as these tumors are often polyphenotypic, with expression of EMA, desmin, GFAP, and/or neuronal markers.

Embryonal tumor with multilayered rosettes (ETMR) may be recognized by the presence of true rosettes and can contain regions of low cellularity with abundant neuropil. On a small biopsy, however, these features may not be well represented (Image 1B). ETMR often harbors amplification of the miRNA cluster C19MC, and this is now considered a defining feature of the entity. A commercially available antibody, LIN28A, is a sensitive, though not entirely specific, surrogate marker for C19MC alteration and can be used as a screening tool. Historical morphologic entities that include ependymoblastoma, medulloepithelioma, and embryonal tumor with abundant neuropil and true rosettes (ETANTR) may harbor C19MC alterations and should be recognized as part of the ETMR spectrum.

Pineoblastomas are embryonal tumors arising in the pineal gland and commonly occur in young people (Image 1C). As with medulloblastoma, the possibility of AT/RT should be considered in the differential diagnosis, and this can be excluded using the INI1/SMARCB1 IHC stain. Pineoblastomas may be associated with germline RB1 or DICER1 alterations.

One may also encounter other embryonal tumors that do not fit the above definitions, justifying the diagnosis of CNS embryonal tumor, not otherwise specified.

Nonembryonal Primary CNS Tumors

Other primary CNS tumors may demonstrate a small round blue cell appearance and are important to consider in the differential diagnosis. Glioblastomas, which are known to have a wide range of histologic patterns (hence the historic name glioblastoma multiforme), may occasionally demonstrate a poorly-differentiated or embryonal appearance. In particular, the 2016 WHO recognizes two histologic patterns that may have a “small round blue cell tumor” appearance: small cell glioblastoma and glioblastoma with a primitive neuronal component. A third recently recognized such category is high-grade glioma with H3F3A G34R/V mutation. These tumors, characterized by a point mutation of the G34 locus of H3F3A, often have broad areas of embryonal morphology and are generally negative for the glial marker OLIG2 (Image 1D). This combination can lead to misdiagnosis as an embryonal tumor. Helpful features, when present, include secondary structures of Scherer such as perivascular, perineuronal, and subpial aggregation of neoplastic cells. These are highly suggestive of an infiltrating glioma and can lead the pathologist to the correct categorization. Immunostaining for G34R/V is available in some laboratories to confirm the diagnosis before definitive testing is performed by sequencing.

Choroid plexus carcinomas may occasionally lose all evidence of papillary architecture and appear as sheets of small round blue cells (Image 1E). Clues to this diagnosis are intra- or paraventricular location of the tumor, young age at diagnosis, and some evidence of nuclear anaplasia. These tumors may show immunoreactivity for EMA, keratin, and S100, but expression patterns are often patchy. INI1/SMARCB1 immunostaining should be performed to exclude AT/RT, which can have areas of papillary architecture. Due to the frequency of sporadic or germline TP53 mutations in choroid plexus carcinomas, IHC for p53 may be helpful but is not specific. Choroid plexus carcinoma is rare overall, but approximately 40% of these tumors occur in the setting of an inherited TP53 loss of function (Li-Fraumeni syndrome). Thus, establishing a diagnosis of choroid plexus carcinoma should prompt consideration for germline testing, and a thorough family cancer history should be obtained.

Hematolymphoid Malignancies

A critical consideration when interpreting a small round blue cell tumor is the possibility of a hematopoietic neoplasm (ie, leukemia/lymphoma). If the specimen is sufficiently large, fresh tissue should be set aside for flow cytometry, and this opportunity can be easily lost if a hematologic malignancy is not initially considered. Radiologically, secondary involvement of the brain by lymphoma or leukemia can take the form of a leptomeningeal or extra-axial mass. In general, for CNS lymphoma to be designated primary, systemic workup should be negative. Primary CNS lymphoma (PCNSL) occurs in older patients (50 to 70 years) and is typically uniformly enhancing on MRI with contrast. Multiple lesions are seen in 25% to 70% of cases. Histologically, PCNSL often displays an angiocentric growth pattern, which can be a helpful clue to the diagnosis. While PCNSL usually shows overt nuclear atypia or anaplasia, more monomorphic lymphomas may be encountered and are

more likely to be confused with other small round blue cell lesions. Immunohistochemically, PCNSL is immunoreactive for CD45/LCA and usually for CD20, as over 95% are of B-cell lineage.

Mesenchymal Neoplasms

Mesenchymal neoplasms are the most common small round blue cell tumors in pediatric oncology. However, CNS involvement by these entities is unusual, and most will only be encountered in the context of metastasis or local invasion by a previously diagnosed neoplasm. Occasionally children may present with primary or metastatic skull or dural lesions without an associated cancer history, so in the context of an extra-axial mass, these entities should be considered. Ewing sarcoma commonly occurs in the extremities or pelvis but occasionally may involve the skull or dura. Immunoreactivity for CD99 (MIC2) should show membrane positivity which is helpful in considering the diagnosis. FLI1 is positive in 85% of cases. Fluorescence in-situ hybridization (FISH) or next-generation sequencing should reveal a rearrangement of the EWSR gene characteristic of this disease. Ewing sarcoma increases in incidence from birth, when it is very rare, to a peak at age 14 years.

Neuroblastoma arises from neural crest cells and primarily affects young children, with a decline to extreme rarity by the age of 7 years. Clinical presentation typically includes catecholamine secretion from the primary adrenal tumor. Clinical behavior is markedly variable with some tumors spontaneously regressing and others rapidly metastasizing. Markers associated with poor prognosis include MYCN amplification and deletions of chromosome 1p. Metastatic neuroblastoma may involve the brain or skull (Image 1F). Routing of tissue for FISH or chromosomal microarray studies is helpful for tumor characterization and risk stratification. The peripheral derivation of neuroblastoma can be demonstrated by immunoreactivity for PHOX2B.

Rhabdomyosarcomas are known to occur in the cranium and are relatively more frequent in the sinuses and skull base. These are generally destructive and rapidly-progressing tumors identified in the first decade of life. The histologic appearance of embryonal rhabdomyosarcoma includes alternating hypo- and hypercellular areas with primitive cells showing features of myogenic differentiation, including the characteristic “strap cells” which have visible cytoplasmic cross-striations. Alveolar rhabdomyosarcomas are more uniform and dense with a prominent collagenous matrix. For both subtypes, myogenin and MyoD1 are useful immunostains reflecting their myogenic lineage. Provision of fresh tissue or touch preparations for FISH can be a helpful adjunct to diagnosis; FOXO1 rearrangements due to gene fusions (PAX3-FOXO1 or PAX7-FOXO1) are a hallmark of alveolar rhabdomyosarcomas and are associated with a poor prognosis.

Some mesenchymal tumors arising in adults may also show a small round blue cell appearance. Solitary fibrous tumor/hemangiopericytoma (SFT/HPC) accounts for <1% of primary CNS tumors and can occasionally display undifferentiated, small cell cytology and a biphasic pattern with paucicellular and hypercellular areas. SFT/HPC frequently shows nuclear immunoreactivity for STAT6, reflecting the NAB2-STAT6 fusion present in the majority of these tumors.

Pitfalls in Diagnosis of Small Round Blue Cell Lesions

A relatively common diagnostic error at the time of frozen section interpretation can arise from sampling of the normal cerebellar cortex. Since the cerebellar granule cell neurons themselves are small, dark, and densely packed, this element of the normal cortex can be mistaken for a neoplastic process. Freezing can also introduce artifactual nuclear atypia, making interpretation even more difficult. Smear preparation can be very helpful in this context, as the granule cell neurons will reveal themselves to be very uniform and hyperchromatic, set in a fine neuropil background, and occasionally will display rosette formation (Image 2).

CSF samples obtained in neonates may occasionally contain small fragments of residual germinal matrix, particularly if the ventricles have been disrupted by placement of a shunt catheter. Germinal matrix cells have a high N:C ratio and finely-dispersed chromatin and may be found in small aggregates. The clinical context of a premature neonate should prompt consideration of this diagnosis. Care must be taken when interpreting these cytologic specimens so as not to misdiagnose an embryonal neoplasm (Image 3).

• The term “small round blue cell” describes tumors with a primitive or undifferentiated appearance and can be applied to a broad spectrum of neoplasms of different cell lineages. Maintaining a broad differential diagnosis will allow the pathologist to triage tissue appropriately for ancillary testing at the time of intraoperative handling.
• The most frequently encountered small round blue cell tumors in the CNS include metastatic small cell carcinoma, lymphoma, and glioblastoma in adults and medulloblastoma, AT/RT, and other embryonal tumors in children.
• IHC for cell lineage-specific proteins (eg, GFAP, cytokeratin, CD45, myogenin, HMB-45) can assist in the workup of challenging small round blue cell lesions.
• CNS embryonal tumors are more common in children, and AT/RT should be considered especially in very young patients. Liberal use of INI1 IHC is encouraged in working up most undifferentiated small round blue cell tumors and is required for all pediatric embryonal tumors.
• Embryonal tumor with multilayered rosettes (ETMR) is characterized by C19MC alteration and includes morphologic entities previously categorized as ependymoblastoma, medulloepithelioma, and embryonal tumor with abundant neuropil and true rosettes (ETANTR). LIN28A IHC is a surrogate marker for the C19MC alteration.

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