A Large Cerebellar Mass.
March, 2004, Case 403-2. Home Page

Shibo Li, M.D., Ph.D. ¹, Kar-Ming Fung, M.D., Ph.D.² First posted on May 1, 2004.

¹ Department of Pediatrics and ² Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma

Kwok Ling Kam, M.B., B.S., FRCPA ¹, Kar-Ming Fung, M.D., Ph.D.² Last updated on on May 25, 2020.

¹ Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois

² Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma

Clinical information: Click thumbnails to see pictures.

The patient was admitted to our hospital because of a large cerebellar mass that was diagnosed in an outside institution. A surgery was performed. The followings are the representative photos of this mass.

A.
Squash B.
Frozen C. D. E. F.

G. H. I.
Vimentin J.
EMA K.
GFAP L.
Cytokeratin

M.
S100 N.
22q deletion FISH

Pathology of the case:

Age of the patient:

The finding that helps to determine the age of the patient is the external granular layer (arrow) in Panel F. This layer is well illustrated in Panel G and H. There are four layers in the cerebellar cortex during this stage of development: the external granular layer, the molecular layer, the Purkinje cell layer, and the internal granular layer.
During formation of the cerebellum, there is an early migration of a population of precursor cells from the neuroepithelium, the premigratory granule cells, to form a second proliferative zone on the outer surface of the cerebellar cortex. This layer is known as the external granular layer and give rise to at least four different classes of neurons: stellate cells, granular cells, Golgi cells, and basket cells.
The external granular layer first appears in the 9^th week of gestation and covers the whole surface by the 14^th week. The maximum thickness is reached at the 24^th week of gestation. The external granular layer is still present at the time of birth and is about 5-6 layers thick. In most babies, this layer is reduced to a single layer by 9 month of age and disappears entirely by 24 months of age. It is important not to mistaken the external granular layer as tumor cells or inflammatory cells.
The cells of the external granular layer migrate inward to form the internal granular layer. The Purkinje cells, however, is originated from the ventricular germinal zone. During the 20^th to 32^th week of development, the cerebellar cortex is transformed from a 4-layer to a 5-layer structure by the transient appearance of a conspicuous hypocellular zone between the Purkinje cell layer and the internal granular layer that is known as the lamina dissecans [Rakic P & Sidman RL, 1970]. Maturation of the cerebellum is earlier along the midline (paleocerebellum) than the periphery (neocerebellum). Therefore, the lamina dissecans disappears in along the midline followed by the periphery. These features can be used for dating of human cerebellum [Friede RL 1973]. This lamina dissecans is not present in this specimen. Click on the following images to see the lamina dissecans (delineated by the arrows on Panel 2). These images are taken from a younger fetus.


1. Lamina dissecans Low-magnification	2. Lamina dissecans High-magnification

In this case, the external layer is present and is about 4-6 cells thick and the lamina dissecans is not seen. It is most likely obtained from a newborn or slightly older than a newborn. In reality, the age of this patient at the time of presentation was three-month-old. Compare the thickness of the external granular layer of Panel G and Panel 2. You can see that the one in Panel G has less layers of cells and is not as densely packed. In contrast, the molecular in the older cerebellum (Panel G) is thicker and less cellular in comparison to the younger cerebellum (Panel 2).
Bonus question: There is another lamina dissecans in primate brains. Where is it? Reference, Reference

Intraoperative consultation:

Panel A is the cytologic preparation. The tissue is composed of pleomorphic cells with moderate to large amount of amphophilic to eosinophilic cytoplasm. There is substantial variation in nuclear size of about 4 times and nucleoli are common and prominent. The cells lack the elongated cytoplasmic processes that are commonly seen in glial tumors (arrow in Panel 3). When the cytologic features are interpreted in conjunction with the clinical information, the most consistent diagnosis is atypical teratoid/ rhabdoid tumor (AT/RT).
Panel B is the frozen section. The tumor is composed of medium- to large-sized tumor cells with pleomorphism and nucleoli. Some of the tumor cells have large amount of cytoplasm and eccentrically located nuclei (arrow) and these are the rhabdoid cells.
Comment: It is almost a rule that the cytologic and nuclear morphologies are often not as well preserved in frozen sections in comparison with cytologic (squash) preparations. In our opinion, cytologic preparations are often very useful to separate AT/RT, ependymoma, and medulloblastoma in pediatric patients (Panel 4). The frozen section, however, provides invaluable information on architecture.

	Pilocytic astrocytoma, cytologic preparation. Note the long cytoplasmic processes (arrow). These processes are typically seen in glial neoplasm in particularly the lower grade ones. Note the lack of prominent nucleoli also.
3. Pilocytic astrocytoma
	Medulloblastoma, cytologic preparation. Note the lack of prominent nucleoli in comparison to AT/RT in Panel A. One must be alerted that anaplasic/large cell medulloblastomas do contain prominent and may be difficult to be separated from AT/RT with minimal rhabdoid changes.
4. Medulloblastoma

Permenant sections, immunohistochemistry:

Hematoxylin-eosin stain: The tumor is highly cellular as illustrated in the medium magnification in Panel C. There is a rich vascular supply. Small necrotic foci are present and are not shown here. On higher magnifications as illustrated in Panels D and E, there is substantial nuclear pleomorphism and variation in cell size. Mitotic figures are common. A subpopulation of the cells has substantial amount of eosinophilic cytoplasm and often prominent nucleoli. Some of them have eccentrically located nuclei as demonstrated in the cytologic preparation and some of these cells are multinucleated (arrow in Panel E). Retrospectively, the rhabdoid features are best visualized in the cytologic preparation. These features are highly consistent with an AT/RT.
Immunohistochemistry: The tumor cells should show loss of INI1 nuclear staining which is the hallmark of AT/RTs (not available in 2004). The tumor cells are uniform and strong positive membranous and cytoplasmic staining for vimentin (Panel I) and EMA (Panel J). Many tumor cells are also positive for glial fibrillary acidic protein (GFAP), cytokeratin (clone AE1/AE3) and S-100 protein as illustrated in Panels K, L, and M, respectively. The tumor cells are negative for desmin.

Cytogenetics:

A piece of fresh tumor tissue was sent for cytogenetics studies at the time of frozen section because of the high suspicion of AT/RT. Routine chromosomal analysis revealed that all twenty cells had apparently normal karyotype. No consistent structural or numerical changes were found. Fluorescence in situ hybridization (FISH) analysis utilizing the BCR gene on 22q11.2 region was also performed, no deletion of 22q11.2 region was detected. However, on interphase FISH of paraffin sections utilizing the same probe as illustrated in Panel N, only one hybridization signal of the BCR gene at chromosome 22q11.2 (green dot) could be detected but the control ABL gene had two dots (red dots). These findings indicated a heterozygous deletion of chromosome 22 involving at least 22q11. 2, which is commonly associated with AT/RT.

Comment:

AT/RTs show polyphenotypical differentiation and often contain non-rhabdoid components, including morphologies that show neuroectodermal, epithelial and mesenchymal differentiations. The proportion of rhabdoid cells can vary from a few to abundant. In cases with rarity of classic rhabdoid cells, a high index of suspicion and thorough search of the rhabdoid are important. Even with absence of rhabdoid cells, small blue cell tumors in the central nervous system of young children (especially those younger than 2 years old) should prompt investigation to rule out AT/RT. Immunohistochemistry, cytogenetics and molecular testing are very helpful.
When this case was diagnosed in 2004, immunohistochemistry for INI1 was not available and therefore the diagnosis was confirmed by FISH.

DIAGNOSIS: Atypical teratoid rhabdoid tumor (AT/RT), WHO grade IV/IV.

Discussion: General Pathology Molecular pathology AT/RT and other rhabdoid tumor Differential diagnosis

General Information

Atypical teratoid/rhabdoid tumor (AT/RT) is a malignant primary childhood brain tumor, WHO grade IV as per the 2016 WHO classification of brain tumors. Malignant rhabdoid tumor was first used in 1981 by Haas et al. to describe a highly aggressive childhood renal tumor with cytologic features suggestive of rhabdomyosarcoma [Haas JE et al., 1981]. Bonnin JM et al. described the association of rhabdoid tumors arising in the brain and kidney in children in 1984. According to Weeks et al., about 13% of children with renal rhabdoid tumors developed brain tumors [Weeks DA et al., 1989]. The first rhabdoid tumor of the central nervous system was described by Biggs et al., in 1987. In common to other malignant rhabdoid tumors arising in children and adults, they contain rhabdoid cells as its salient histologic feature.

AT/RT often contains non-rhabdoid components with medulloblastoma-like components and sometime malignant and neoplastic epithelial and mesenchymal components. Rorke et al. published the first comprehensive series of 32 cases on rhabdoid tumors of the central nervous system and also used the term atypical teratoid/rhabdoid tumor (AT/RT) to describe these tumors [Rorke LB et al., 1996] as an attempt to emphasize the diverted histopathologic features in these tumors. Deletion and mutation of the SMARCB1 gene (synonyms: INI1, SMARCB1, hSNF5) in chromosome 22q11.2, and very rarely SMARCA4 which codes the BRG1 protein, are the molecular defining features of AT/RTs [Biegel et al., 2002]. A cell line has been established [Yachnis AT et al., 1998].

AT/RT is an uncommon tumor comprising about 2% or less of all childhood brain tumors and is less frequently seen than medulloblastoma. There is a slight male predominance and most of the tumors occur under the age of two and only rare examples has been reported in adults [Luttenbach J et al., 2001]. Similar to other malignant tumors of the central nervous system, they rarely metastasize outside the central nervous system [Guller E et al., 2001]. The posterior fossa and the cerebellar pontine angle are the most common place of occurrence with a combined incidence of 49% (38% in cerebellum, 11% in cerebellar pontine angle). AT/RT has a distinct tendency to invade into the surrounding tissue in the cerebellar pontine angle. The cerebrum harbors the other 28% of tumors and other uncommon sites including pineal, brainstem, suprasellar, and spinal cord are primary locations of the rest of the tumor (13%) [Rorke LB et al., 1996]. Disseminated tumor is seen in about one third of the cases at presentation. The prognosis is poor [Rorke LB et al., 1996; Burger PC et al., 1998; Bambakidis NC et al., 2002], with a 3-year survival of 22% from the Germany HIT trial center [Biggs et al., 1987]. High dose chemotherapy and radiation can potentially prolong the survival [Meyers SP et al., 2006; Lafay-Cousin L et al., 2012; Geyere JR et al., 2005; Chi et al., 2009]. Rare prolonged survivals have been reported [Hirth A et al., 2003; von Hoff K et al., 2011]. Molecular subgroups of AT/RTs have also been identified using global and transcriptional analysis and the data may help to stratify patients into different risk groups [Torchia J et al., 2006]. MRI findings include variably contrast enhancement, with hyperintensity on FLAIR images and diffusion restriction, the latter is indicative that the tumor is highly cellular [Howlett DC et al., 1987; Meyers SP et al., 2006].

Pathology

Rhabdoid cells are medium sized to large, oval to round cells. In the most classic example, the rhabdoid cell contains an eosinophilic, hyaline-like cytoplasmic globule resembling an inclusion body and occupies a large portion of the cytoplasm. This hyaline globule displays the nucleus to an eccentric location. The less classic examples contain a substantial amount of eosinophilic, often hyaline-like cytoplasm and eccentrically located nuclei. Although the morphologic features are suggestive of a rhabdomyosarcoma, cytoplasmic striations typical for rhabdomyoblastic differentiation should not be present. Marked pleomorphism is seen in most cases and nucleoli are usually prominent. Multinucleated giant cells are common. Mitotic figures and atypical mitosis are common. The degree of necrosis is variable but may not be extensive.

Only about 13% of AT/RTs are composed exclusively of rhabdoid cells. Non-rhabdoid components are seen in the rest. About two third to three quarter of AT/RTs are associated with medulloblastoma or primitive neuroectodermal tumor (PNET) like components. Neoplastic epithelial components may occur as adenocarcinoma-like components, squamous cell with keratinization, or just simply epithelial cells arranged in nests. Neoplastic mesenchymal components can also be seen. The amount of rhabdoid cells can vary greatly from uncommon to substantial. AT/RT with substantial medulloblastoma/PNET like component may be mistaken as medulloblastoma or PNET [Burger PC et al., 1996]. Under the electron microscopy, rhabdoid cells contain bundles of tightly packed intermediate filaments arranged in whorls.

Immunohistochemistry, the defining diagnostic staining is loss of INI1 nuclear immunoreactivity in the tumor cells. The classic rhabdoid cells are also strongly and uniformly positive for vimentin. In fact, immunohistochemistry for vimentin is helpful in identifying rhabdoid cells. Other than vimentin, a long list of antigens is variably detected in rhabdoid tumors reflecting the polyphenotypic nature of these tumors. Epithelial membrane antigen (EMA) is detected in the rhabdoid cells and epithelial components. About half of the cases are positive for smooth muscle actin. Expression of these two antigens are rather unusual for other tumors of the central nervous system. S-100 protein is variably detectable among different cases. Intermediate filaments including neurofilament, glial fibrillary filaments, and cytokeratin are detectable in many cases. Synaptophysin is detectable particularly when a medulloblastoma-like component is present. Placental alkaline phosphatase (PLAP) and beta-human chorionic gonadotrophin (hCG) are not detectable but alpha fetal protein (αFP) can be demonstrated in some cases [Rorke LB et al., 1996; Burger PC et al., 1996].

Molecular pathology:

Biallelic inactivation of the SMARCB1 gene (INI1/hSNF5) located in chromosome 22q11.2, either by homozygous deletion or having one mutant allele and the other allele with deletion or mitotic recombination, accounts for majority of molecular alterations seen in AT/RT [Rorke LB et al., 1996; Biegel JA et al., 1992, Rorke LB et al., 1995; Biegel JA et al., 1999; Biegel et al., 2002; Taylor MD et al., 2000]. Such deletion is also detected in other malignant tumors such as epithelioid sarcoma and renal medullary carcinoma. Detection of 22q11.2 deletion by interphase fluorescence in situ hybridization (FISH) would be a helpful aid to establish a diagnosis [Bruch LA et al., 2000]. Very rarely, mutation or inactivation of the SMARCA (BRG1) gene which also belongs to the SWI/SNF pathway can be seen in AT/RT. Molecular confirmation can be done using sequencing (such as next generation sequencing), FISH and chromosomal microarray analysis.

SMARCB1 (INI1/hSNF5) is a member of the SWI/SNF chromatin-remodeling complex that functions in a reversible manner to remodel nucleosomes (i.e. chromatin remodeling) forming a closed and native state to an open and active state [Biegel JA et al., 2002; Schnitzler et al., 1998]. SMARCB1 (INI1/hSNF5) probably functions as a tumor suppressor gene but its molecular pathway and functions within the SWI/SNF complex have not been fully revealed. Germ line mutations of the SMARCB1 (INI1/hSNF5) and SMARCA4 genes have been reported to greater than 1/3 of the cases [Hasselblatt M et al., 2014; Biegel JA et al. 1999; Taylor MD et al., 2000; Sevenet N et al., 1999], and are seen in rhabdoid tumor predisposition syndromes 1 and 2, molecular genetic studies should be done in all new cases. Malignant rhabdoid tumors arising in more than one anatomic site are common in patients with germline mutations. Genetic aberrations of INI1/hSNF5 have also been currently described in choroids plexus carcinoma [Sevenet N et al., 1999; Wyatt-Ashmead J et al., 2001].

Recently, meta-analysis shows 3 distinct molecular subgroups of AT/RT, including ATRT-TYR, ATRT-SHH and ATRT-MYC [Ho B et al., 2020] using data based on gene expression array profiling and methylation profiling [Johann PD et al., 2016; Capper D et al., 2018; Schwalbe EC et al., 2017]. Each of these subgroups have their clinical and molecular characteristics, including age of onset, tumor location, SMARCB1 alterations and oncogenic pathways.

AT/RT and other rhabdoid tumors:

Tumors with malignant rhabdoid phenotype, irrespective of their location and organ being involved and the age of the patients, behave in a highly aggressive manner and carry a grave prognosis. Malignant rhabdoid tumors are most frequently seen in infants and children and are predominantly restricted to the kidney, the central nervous system (AT/RT) and soft tissue. In adults, malignant rhabdoid differentiation has been described in many different organs and often occurs as a component arising in a pre-existing neoplasm including carcinomas [Chetty R, 2000; Kuroda N et al., 2000; Parham DM et al., 1994], sarcomas [Parham DM et al., 1994; Oshiro Y et al., 2000; Morgan MB et al., 2000], melanocytic tumors [Borek BT et al.,1998; Chang ES et al. 1994], or other neoplasms.

In contrast, pediatric malignant rhabdoid tumors are often associated with deletion or mutation of INI1/hSNF5 gene. Pediatric malignant rhabdoid tumors arise mainly in the kidney. The more common extrarenal sites are the soft tissue and central nervous system (i.e., AT/RT). Pediatric rhabdoid tumors arising in the kidney and soft tissue are often of pure rhabdoid phenotype and are distinct from composite type arising in adults. AT/RTs share the feature of adult malignant rhabdoid tumors in having non-rhabdoid component but also feature of pediatric malignant rhabdoid tumor in the high frequency of deletion or mutation of INI1/hSNF5 gene.

Differential diagnosis

Although rhabdoid phenotype has been described in tumors of including meningioma [Perry A et al., 1998] and glioblastoma [Lath R et al., 2003], the rhabdoid phenotype is not a frequently encountered histologic pattern in primary tumors of the central nervous system other than AT/RT. These reported cases occurre in adults. Classic features of the “mother tumor” are usually found after careful search. The age of the patient is of great importance as only very few AT/RTs occur in adults. Before a diagnosis of AT/RT is made, the absence of a renal or extrarenal-extraneural rhabdoid tumor must be confirmed. The occurrence of two rhabdoid tumor can represent multifocal tumor or metastasis. As discussed earlier, patient with more than one malignant rhabdoid tumor in different locations may have germ line mutations.

AT/RT with substantial proportion of embryonal tumor component can easily be misdiagnosed [Burger PC et al., 1998]. Rhabdoid cells can usually be found after careful search. One of the key features is the presence of at least distinct if not prominent nucleoli. The rhabdoid change may not be as impressive or dramatic as it appears on text books. A high index of suspicion, immunohistochemical staining with INI1, as well as molecular and cytogenetic studies should be done in suspicious cases, and should be done in all embryonal-looking brain tumors in patients younger than 2 years old. AT/RT merits separation from medulloblastoma and PNETs for its grave prognosis

AT/RT with significant epithelial component can be mistaken as metastatic carcinoma. Again, age and clinical history is very helpful as metastatic carcinoma is extremely rare in infants. The pathologic features are also different.

AT/RT may show resemblance of choroid plexus carcinoma. Both of these tumors occur in young infants and with cerebellar pontine angle a common site. To make this issue more complicated, deletion of chromosome 22q11.2 has also been described in some malignant childhood tumors that are reported as choroids plexus carcinoma [Sevenet N et al., 1999; Wyatt-Ashmead J et al., 2001]; however, some authors believe that they may represent intraventricular AT/RT instead. On the other hand, about 40% of choroid plexus carcinoma shows germline TP53 mutation [Tabori U et al., 2010], and may be the first manifestation in patients with Li-Fraumeni Syndrome.

Germinoma and other germ cell tumors can be easily distinguished from AT/RT. Although germinoma has large cells with large nuclei and prominent nucleoli, it also contains significant amount of lymphocytes sprinkling around blood vessels which is not usually seen in AT/RT. Immunohistochemistry by INI1 should solve the puzzle. Besides, germ cell tumors occur predominantly along the midline in the pineal or sellar/suprasellar regions which are uncommon sites for AT/RT.

Related cases:


A. Squash	B. Frozen	C.	D.	E.	F.

G.	H.	I. Vimentin	J. EMA	K. GFAP	L. Cytokeratin

M. S100	N. 22q deletion FISH