Squash Cytology of Paediatric CNS Tumours

Presented by – Dr. V. Nagesh Kumar (1st Yr PG)

Chair Person: Dr. Kumuda (Prof)

Moderators: Dr. Rama Raju (Assoc Prof)

Dr Shrinivas (Asst Prof)

Dr. Harvey Williams Cushing (1869 – 1939) Dr. Louise Eisenhardt

(1891 – 1967)

Introduction

• Central nervous system (CNS) is one of the most challenging domains for the neurosurgeon and Pathologists

• The role of intraoperative pathological diagnosis is crucial in neurosurgery

Introduction

• Making a diagnosis of CNS lesions is difficult on the basis of clinical and radiological findings only

• Cytological and/ or histological diagnosis is required for confirmation and proper management

Introduction

• Besides rapid decision making during neurosurgical procedures, it is also to be ensured that minimum injury is caused to the normal brain structures surrounding the intracranial neoplasm

Introduction

• In this regard, imprint cytology or squash smear prepared from small biopsy during surgery seems to be very helpful for the operating surgeon to make a decision on further management

Introduction

• The application of smear techniques as a means of obtaining rapid diagnosis for neurosurgical biopsies was first advocated by Dr Eisenhardt and Dr Cushing in the USA in early 1930

• Since then, although the technique has been modified by various individuals by changing the fixative or stain, the basic principle has remained unchanged

Introduction

• Though this technique receded to the background with the advent of CT guided stereotactic biopsy, it has regained importance due to its technical simplicity

• Three simple techniques allow rapid examination of biopsy material during surgery: touch preparations, squash preparations, and the smear

Squash Preparation

• Tissue is crushed between two slides and then stained

• Similar to touch preparations, this method will give excellent cytology, although some cells may show some crush artifacts

Squash Preparation

• As the tissue spreads out under the force produced by the two glass slides, its structure partially breaks down, thereby releasing more cells

• Notably, neoplastic cells that elaborate their own matrix will retain their relationship to this milieu on the slide

Examination of the smear

• A multilevel process and entails observing at every level

– Radiology– gross examination, and – how the tissue physically separates

Examination of the smear

• After it is stained, hold it up to the light What does it look like? – Is it pink or blue?– Lmpy or smooth?– Easy to spread or resist spreading?

Examination of the smear

• Under the microscope– Intermediate magnification

• the lesion's matrix becomes clear: glial, liquid, epithelial, or cohesive

• Finally, at high power – the cells reveal their identity– Their cytoplasm generally indicates the cell type,

whereas their nuclei tell whether the process is reactive or neoplastic, benign or malignant

Biopsies of Gray Matter

• Produce smooth, relatively pink smears

• They are hypocellular, which distinguishes them from tumors and inflammatory disorders

• A few delicate capillaries often stretch around the smear, whereas larger vessels are dragged off to the end of the slide

Biopsies of Gray Matter

• Neurons and their synapses make up the essence of gray matter

• Fine granularity of gray matter or neuropil depends on the multitude of axons synapsing on dendrites

Normal gray matter

Biopsies of White mater

• Normal white matter lacks the neurons and neuropil of gray matter

• It contains two main populations of cells that can be difficult to distinguish:– Oligodendrocytes - a dense, small, round,

blue nucleus– Astrocytes - larger, slightly round nucleus with

more open chromatin

• Unless you see some defining cytoplasm, you cannot reliably distinguish these nuclei. Rather, you identify a cell by the company it keeps, by its context in the surrounding tissue

A. Low magnification view showing the light fluffy appearance of slightly gliotic white matter.

B. Intermediate power showing a reactive astrocyte (arrow) in a spattering of small, round, blue cells. Although many of these other cells are most likely oligodendrocytes (arrowheads), they cannot be reliably distinguished from a few infiltrating lymphocytes or nonstimulated macrophages.

Cerebellar cortex

• In a smear, identifying cerebellar cortex requires – recognizing the monotony of its granular

neurons – finding a fine neuropil background, and – looking for the occasional, helpful Purkinje

neuron

Normal cerebellar cortex

• Distinguishing normal cerebellum from a small, round, blue cell tumor can be tricky

• However, normal cerebellum smears evenly, like all other brain, and the confounding internal granular neurons are oppressively monotonous at high power

Smear Patterns

• Noncohesive smears

• Cohesive smears

• Epithelial tissue smears

• Glial tissue smears

• Necrotic debris smears

Noncohesive smear

Noncohesive tissues. In noncohesive processes, cells grow without significant intercellular attachments. Their matrices are weak and are insufficient to bind the cells together.

Noncohesive smear

This is a classic smear from a pituitary adenoma

Cohesive smear

Several different types of tissues, including some tumors and dura, are tightly bound together by either dense reticulin or collagen bands. These tissues are unwilling to give up their cells without a fight.

Cohesive smear

Smear of a cohesive schwannoma

Epithelial tissues come in different forms, but all share two characteristics: they are bound to each other by cell surface connections and they have distinct cell membranes. On a smear, squamous tissues form sheets (upper cells). Tubular epithelium frequently forms balls (lower cells). Papillary epithelial tissues form papillary structures on smears.

Epithelial tissue smear

Epithelial tissue smear

Metastatic lung carcinoma

Glial tissue smear

A smear prepared from gliotic tissue, be it reactive or neoplastic, will reflect these highly intermingled processes. At low magnification, it will look like puffs of cotton seeds clumped around strands of cotton fibers (center). On high power, reactive or neoplastic astrocyte nuclei connect directly to the glial matrix they have elaborated (right).

Glial tissue smear

Moderate reactive gliosis near a metastasis

Glial tissue smear

High-power view of a reactive astrocyte in this smear

Necrotic debris smear

This fragment of tissue from a glioblastoma is necrotic. The pale, dead nuclei (arrows) are diagnostic evidence of necrosis. This sample also has some blood bound within it. Remember, erythrocytes can lyse during the fixation step and leave only ghosts, which can look like dead nuclei.

Paediatric CNS Tumours

Paediatric CNS Tumours

• most common solid organ tumor in children

• are second only to leukemias in children

• 70% of pediatric brain tumors are in the posterior fossa– Pilocytic astrocytoma– Medulloblastoma– Ependymoma

Paediatric CNS Tumours

• Primary

• Secondary– Metastatic– Local invasion

• Tumors of the spinal cord

Paediatric CNS Tumours

• Most common types– Astrocytomas

• Grades I-IV

– Medulloblastomas • primitive neuroectodermal tumor-PNET

– Meningiomas– Pituitary adenomas

Infratentorial or Posterior Fossa

• Astrocytomas

• Medulloblastomas

• Atypical teratoid/rhabdoid tumors

• Ependymomas

• Choroid plexus tumors

Parasellar

• Astrocytomas

• Craniopharyngiomas

• Germ cell tumors

Medulloblastoma• Embryonal tumor of the cerebellum and at

sites throughout the neural axis showing primitive neuroectodermal cells with neuronal differentiation

• considered malignant, grade IV neoplasms because of their potentially rapid growth and to seed distant sites of the nervous system through the ventricles and subarachnoid spaces

Medulloblastoma

• common solid tumor of children and young adults

• The classic form has neuroblastic rosettes (tumor cell nuclei around cytoplasmic processes)

• Mitoses and apoptoses may be frequent

MRI – T1, T2 & gad enhanced images of Medulloblatoma

In a smear examined at medium to high microscopic powers, classic medulloblastomas show cells as if glued end-to-end, small numbers of nuclei stick together, forming short chains or circles, these represent true rosettes

Medulloblastoma smears at medium power. Few nuclei remain fully isolated from other nuclei (white arrowheads). Instead, many of the cells seem to form short chains or clusters (black arrowheads) and some form larger, circular arrangements.

Features of a classic medulloblastoma: A.Cells with scant-to-no cytoplasm having nuclei displaying salt-and-pepper chromatin but no nucleolus. Nuclear molding (arrows) reflects cellular cohesion in the presence of minimal cytoplasm. B.Occasional circles of cells (arrowheads) appear to be more than a fortuitous arrangement of cells, because many of their nuclei mold onto each other and lumen they form has a distinct edge.

Medulloblastoma showing neuroblastic differentiation

Desmoplastic Medulloblastomas

• Like all variants these smear easily

• displays abundant pink neuropil

• Relatively monotonous nuclei dispersed in a fine neuropil matrix

• Minimal cytoplasm and has more homogenous nuclei than classic medulloblastomas but retains the salt-and-pepper chromatin

Desmoplastic medulloblastoma

• This tumor displays a prominent nodular growth pattern on permanent sections. These nodules are not really desmoplastic lumps surrounded by fibrous tissue but rather islands of better-differentiated neural cells within neuropil that are demarcated by poorly differentiated bands of cells

Atypical teratoid rhabdoid tumors (ATRT)

• ATRTs are a tumor of early childhood(<2 years of age) and rare after age 4 years

• These tumors are neither teratomas nor do they produce muscle

• ATRTs lack neuronal differentiation

• Easily be confused with medulloblastomas

• Have predilection for the cerebellum and posterior fossa, they can occur anywhere along the neural axis

• These tumors characteristically have lost one chromosome 22; monosomy 22, in the right clinical setting, is considered diagnostic of an ATRT

• Unlike medulloblastomas, which can be eradicated in many patients, these are highly malignant tumors for which no satisfactory treatment has been developed

Atypical teratoid rhabdoid tumor neuroradiology

Atypical teratoid rhabdoid tumorA.The field view of this microscopic slide shows a choppy or rough spreading of the tumor. B.Among the cellular clumps at low magnification, tumor cells shed easily. They accumulate in large numbers around stiffened or rigid blood vessels (arrow). Notice the tiny piece of involved cerebellum that was part of the tissue sampled (cb, demarcated by lines); this piece had been invaded by tumor but still retained some of its cellular components.

Vascular predilection of the tumor. In both preparations, cells cluster around vessels. In addition, the vessel walls appear thickened and the endothelial cells enlarged (B).

Multinucleated giant tumor cells are helpful, when present. Their nuclei match those of the surrounding mononuclear tumor cells.

ATRTs may have subclones displaying epithelioid features: large cells having abundant, eosinophilic cytoplasm, sharp cell borders, and eccentric nuclei

ATRT (Higher magnification)A.Collection of primitive cells having scant cytoplasm and a salt-and-pepper chromatin (arrowheads). Notice how an occasional cell having a similar nucleus can also have more abundant and distinct cytoplasm (arrow). B.The volume of cytoplasm ranges from scant to abundant (arrow). As cytoplasmic volume increases, the nuclei remain eccentric rather than central.

C. A true rhabdoid cell, rather than just an epithelioid cell with an eccentric nucleus, should have condensed cytoplasm and perhaps fine internal filaments or whorls (arrow). D. This multinucleated cell could really be a very tight clustering of smaller epithelioid cells, because some distinct cytoplasmic borders are present (arrowhead); this is not a cell of a medulloblastoma.

Choroid plexus tumors

• Occurs in any portion of choroid plexus, but usually as papillary neoplasms of lateral ventricle of children and fourth ventricle of adults

• Uncommon (0.4 to 0.6% of intracranial neoplasms)

Choroid plexus papilloma

• Grade I of IV - benign

• Rare (<1%), slow growing tumor

• commonly in ventricular system and associated with hydrocephalus

• Often causes developmental delay, behavioral problems or epilepsy in children

• 85% occur at age 10 years or less; often present at birth

• Needle biopsy not recommended since histologically resembles normal choroid plexus

• 10-30% become histologically malignant

• High survival unless becomes malignant (then 5 year survival is 26%), although histology does not predict behavior

Well-demarcated intraventricular (or cerebellopontine angle) mass with hydrocephalus. Calcification especially frequent in fourth ventricular tumors. In adult patients the fourth ventricle and in children third ventricle is more common. The tumor is attached to the choroid plexus.

Choroid plexus papilloma (transverse section of the brainstem and the cerebellum). Pinkish, granular growth with lobular contour in the lateral recess of the fourth ventricle, at the cerebellopontine angle.

A. In the low-power smear, the tumor has grapelike papillary clusters of cells bulging off vessels. B. A cross-section transforms these clusters into dendritic arbors or fronds of tumor growing on fibrovascular cores.

C. The tumors shed bland epithelial balls or shells of cells. D. Permanent sections show the labyrinth of tumor cells lining vascular stalks.

E. At high magnification, the tumor nuclei in individual shells are bland and monomorphic. F. Histological sections show the nuclei to be more hyperchromatic, heaped-up, and longer than those of normal choroid plexus.

Choroid plexus papilloma smears. A.At intermediate magnification, sheets of cells stripped from papillae form a honeycomb pattern. B.High magnification shows distinct cytoplasmic borders separating the cells in these sheets.

C. Cells further sheared off the sheets retain their polarized epithelial features; they have distinct cell membranes and eccentric bits of cytoplasm. D. Ribbons of epithelial cells mirror the fibrovascular structures on the permanent sections.

E. Because they are rigid, thick, and refringent, psammoma bodies often jump out of the grapelike clusters. F. At high magnification, isolated psammoma bodies display their diagnostic and aesthetic concentric spheres or laminations.

Choroid plexus carcinoma

• WHO grade III of IV

• Extremely rare and resembles metastatic carcinoma but usually occurs in children

• Associated with germline p53 mutations

• Positive stains: EMA, keratin, S100, INI1; GFAP (20%)

• DD: atypical rhabdoid tumor (INI1 - ve)

Left: Contrast-enhanced T1-weighted MR images showing a choroid plexus papilloma that developed in the third ventricle. The vascular tumor pedicle arising from the choroid plexus and the associated hydrocephalus are typically observed in cases of these lesions.

Right: An MR image showing a choroid plexus carcinoma of the lateral ventricle without hydrocephalus. In children such appearances can be similar to those of an ependymoma or an intraventricular meningioma.

Gross: well circumscribed, brown-red, cauliflower-like mass; variable hemorrhage, necrosis and invasiveness

Choroid plexus carcinoma. (×200; h&e stain) shows a solid growth of markedly pleomorphic cells with loss of any papillary growth pattern.

Craniopharyngiomas• Benign - typically very slow growing tumors

and arise from the cells along the pituitary stalk

• usually suprasellar neoplasm, which may

be cystic, that develops from nests of epithelium derived from Rathke's pouch

• Rathke's pouch is an embryonic precursor of the anterior pituitary

• Resistant to smearing, reveal cohesive sheets of squamous cells and keratinous debris. Background can show cholesterol crystals, calcified debris, foreign body type giant cells and histiocytes.

• Microscopically: – adamantinomatous (pediatric type), – papillary (adult type) – mixed

Spongy and cystic transformation of the cell cords and connective tissue. Massive nodule of compact "wet keratin".

Ramified cell cords composed of squamous epithelium bordered by a layer of cuboidal basal cells. These cell cords are separated by an edematous, loose connective tissue that exhibits cystic change in certain areas.

• DD: – metastatic carcinoma (usually no

calcifications, no squamous epithelium)

– pilocystic astrocytoma (if only gliosis is sampled, more cellular, has microcysts)

– Rathke cleft cysts (CK8+, CK20+; both negative in craniopharyngioma)

Intracranial Germ Cell Tumors• 1-3% of primary pediatric CNS tumors

• Multiple tumor types seen: – Germinomas -55% – Teratomas and mixed germ cell tumors -33% – Others: malignant endodermal sinus tumors,

embryonal cell carcinomas, choriocarcinomas, teratocarcinomas-10%

• In all but germinomas, serum and CSF alpha-fetoprotein (AFP) and βHCG may be elevated

Germinoma

• MC intracranial germ cell neoplasm• Seen in teenagers and young adults• May derive from ectopic rests,

transformation of resident germ cells or migration of germ cells late in development

• MC site is pineal region; also anterior or posterior third ventricle, rarely fourth ventricle

Germinoma

• Relatively good prognosis

• Very sensitive to radiotherapy and chemotherapy

• Metastases may be due to surgical displacement of tumor; spinal cord metastases occur in 10-15% of patients

Large pleomorphic cells with vacuolated cytoplasm ,large nuclei and streaking of lymphoid cells

• DD: – embryonal carcinoma (25% are PLAP+)– Metastatic carcinoma (keratin+, EMA+, 13%

are PLAP+)

Embryonal carcinoma

• Characterized by rapid and bulky growth and spread to liver and lungs; 60% have metastases at presentation

• Prognosis poorer than germinoma

• May be associated with precocious puberty

• Positive stains: alpha-fetoprotein

Yolk sac tumor

• Rare intracranial tumor, usually in pineal or suprasellar regions

• Also called endodermal sinus tumor• Prognosis poorer than germinoma (median survival 2 years

or less)• Gross: usually large• Micro: tubulopapillary structures with vacuolated cuboidal

cells, cystic spaces with eosinophilic hyaline bodies, and Schiller-Duval bodies

• Micro images: various images-solid pattern in pineal gland• Positive stains: alpha-fetoprotein

• Tumor occupies dorsal thalamus, cerebral peduncle, mesencephalic tegmentum and pons with compression of the third ventricle and adjacent structures

Tumor occupies dorsal thalamus, cerebral peduncle, mesencephalic tegmentum and pons with compression of the third ventricle and adjacent structures

 crushed smears showed loose aggregates of medium sized hyperchromatic undifferentiated cells with ovoid nuclei, moderate anisonucleosis, discernible nucleoli, and scant to moderate amounts of cytoplasm

Teratoma

• Tissue derived from ectoderm, endoderm and mesoderm (at least 2 of 3 germinal layers)

• Usually well differentiated / grade I of IV

• Congenital cases are usually fatal

• Mature teratomas: have well differentiated tissue from all three germinal layers

• Immature teratomas: have less differentiated tissue from any of the three germinal layers

• Poor prognosis: tissue resembling medulloepithelioma, neuroblastoma, retinoblastoma or ependymoblastoma

• Pineal teratomas are more common in males, but saccrococcygeal teratomas are more common in females

• Treatment: newborns - complete surgical excision (difficult)

MRIof the head shows a mass measuring 4.2 cm in diameter with cystic contents and small areas of nodular and rim enhancement. The mass causes marked compression of the cortical mantle throughout the inferior parietal and temporal regions.

Choriocarcinoma

• Prognosis poorer than germinoma - median survival 22 months in cases with high hCG levels

• Serum levels of hCG are helpful• Micro: syncytiotrophoblasts (large

multinucleated cells) and cytotrophoblasts• Positive stains: hCG• DD: metastatic choriocarcinoma (from

gonads or placenta)

References

• Diagnostic Neuropathology Smears, 1st Edition by Joseph, Jeffrey T

• Intraoperative neurocytlogy of primary CNS neoplasia, Review article by shama and Deb

• Internet

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