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Symposium
Cytology of soft tissue tumors: Malignant small round cell
tumors
Malignant small round cell tumor (MSRCT) is a group of
morphologically similar neoplasms with variable histogenesis
and biological behavior.[1] This group of tumors with different
origin includes: Neuroblastoma (NB), Ewing’s sarcoma
(EWS)/Primitive neuroectodermal tumor (PNET), malignant
lymphoma (ML), rhabdomyosarcoma (RMS), Wilm’s tumor
(WT), Desmoplastic small round cell tumor (DSRCT). Other
differential diagnoses of MSRCTs include small cell osteogenic
carcinoma and synovial sarcoma.
Differential diagnoses are difficult due to their undifferentiated
or primitive character. Malignant small round cell tumors
are characterized by small relatively undifferentiated cells.
Accurate diagnosis is required for many of these tumors and
imprecise diagnoses, such as small cell tumor, should be
strongly discouraged. This would enable the institution to
implement appropriate therapeutic protocols, including new
adjuvant chemotherapy in advanced malignancy.[2]
A vast majority of MSRCTs are difficult to differentiate using
light microscopy alone. In the last few years, a variety of
ancillary diagnostic techniques such as immunohistochemistry,
electron microscopy, cytogenetics, and molecular genetics
have provided precious tools for addressing this diagnostic
dilemma. The immunocytochemical panel that is useful in
the diagnosis of round cell tumors is shown in Table 1. The
use of ancillary studies has proven to be a decisive step in
fine needle aspiration (FNA) diagnosis.[3] It is well recognised
that immunophenotyping of antigens to identify the tissue
of origin is critical in the diagnosis of MSRC tumors. The
recent molecular genetic characterization of the chromosome
abnormalities characteristic of small round cell tumors has
resulted in greatly improved detection strategies, based
mainly on the techniques of fluorescence in situ hybridization
(FISH) and polymerase chain reaction (PCR).
Ewing’s sarcoma: Ewing’s sarcoma (EWS) is a malignancy
ARVIND RAJWANSHI
Department of Cytology and Gynaecologic Pathology, P.G.I.M.E.R, Chandigarh, India
For correspondence: Dr. Arvind Rajwanshi, Department of Cytology and Gynaecologic Pathology, PGIMER, Chandigarh - 160 012,
Union Territory, India. E-mail: rajwanshiarvind@hotmail.com
Journal of Cytology / July 2008 / Volume 25 / Issue 3
of the bone and soft tissue that is usually seen in the
paediatric population but may also be found in adults. It is
an undifferentiated neoplasm composed of small round cells
lacking any specific differentiation. In the majority of the
cases, abundant cytoplasmic glycogen can be demonstrated
by PAS.[4] FNAC reveals tumor cells that are uniform in size
and shape and that are present singly or in small clusters.
Nuclei are round to slightly irregular with inconspicuous
nucleoli. The cytoplasm has vacuoles and the cells are
arranged in variable numbers of pseudorosettes. Electron
microscopic examination confirms the presence of glycogen
in areas corresponding to cytoplasmic vacuoles. There may be
scattered cell junctions, but usually no cytoplasmic filaments
or neurosecretory granules are seen.[1]
MIC2 is a specific marker for ES and PNET. MIC2 is a ubiquitous
pseodoautosomal gene located on the short arms of both X
and Y chromosomes. The gene product, a cell membrane
protein, is recognized by monoclonal antibody (MoAb) HBA-
71 and MoAb 12E7 and RFB-1.[5] Although a sensitive marker,
MIC2 is not specific and can be seen in some cases of other
small round cell tumors such as RMS, WT, small cell carcinoma
of the lung, and T-cell non-Hodgkin’s lymphomas,[6,7] whereas
neuroblastomas have been found to be universally negative
for MIC2.[8] The utility of FLI-1 expression in EWS/PNET is
specific and useful in differential diagnoses. FLI-1 has been
recently proposed as an additional immunohistochemical
marker of ES/PNET alongside the traditional MIC2.[9]
Approximately 90% of ES/PNET have a specific t(11.22) (24, q
12) translocation that results in the fusion of EWS and FLI-1
genes. Various other translocations have been described that
involve other members of the ETS gene family. The t(21.22)
(q12:q12) translocation involves the ERG gene whereas
t(7:22) t(q22:q12) involves the ETV1 gene. These transcripts
can be detected using molecular techniques such as RT-PCR
and FISH.[10]
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Rajwanshi: Cytology of round cell tumors
Table 1: Immunocytochemical panel useful in the diagnosis of round cell tumors
Antibody EWS/PNET RMS
Cytokeratin - -
Vimentin + +
LCA - -
NSE - -
MIC2 + -
Desmin - +
LCA - Leucocyte comman antigen, NSE - Neuron specific-enolase, EWS - Ewing’s sarcoma, PNET - Primitive neuroectodermal tumor, RMS - Rhabdomyosarcoma, NB - Neuroblastoma,
DSRCT - Desmoplastic round cell tumor
Neuroblastoma: Neuroblastoma (NB) is the most common
malignant tumor of infancy and childhood. The majority (90%)
of the tumors, occur in patients less than ten years of age
and there is a slight male preponderance. The tumor arises
from the undifferentiated precursor cells of the sympathetic
nervous system but the adrenal gland is found to be the seat
of primary growth in about a quarter of the cases. In FNAC
smears, the tumor cells may be disposed singly or arranged
in small clusters. The cells are small and undifferentiated
with a high nuclear cytoplasmic ratio. Small clusters of cells
may be separated by pale blue to light purple fibrillar matrix,
and structures corresponding to pseuodorosettes are seen
in histological sections.[1] A panel of monoclonal antibodies
directed against neuroblastoma has been found to be a
valuable aid to diagnosis. The most widely used antibodies
are neuron specific-enolase (NSE), protooncogene product
9.5 (PGP 9.5), and NB84 which are detected in about 75–80%
of all cases. But NB84 positivity can be seen in other tumors
also such as Ewing’s Sarcoma, squamous cell carcinoma,
and leiomyosarcoma, and NSE positivity can also be seen
in RMS.[11,12]
Neuroblastomas are characterized by the deletion of the
short arm of chromosome 1 (1p 36; 2-3) and amplification of
the protooncogene MyCN abnormality of the chromosome
number. Deletion of 1p is the common cytogenetic
abnormalities. Most (90%) of the cases show the secretion
of catecholamines and therefore, almost all neuroblastomas
produce enzymes such as tyrosine hydroxylase (TH) and DOPA
carboxylase which are the some of the first enzymes in the
catecholamine biosynthetic pathway. RT-PCR can be used to
detect mRNA of TH and DOPA carboxylase and is very helpful
in differentiating NB from other MSRCTs.[13]
Rhabdomyosarcoma: Pediatric soft tissue sarcomas account
for approximately 5% of all childhood cancers and over one-
half of them are rhabdomyosarcomas. They are classified into
two main morphological categories: embryonal (ERMS) and
alveolar (ARMS).[14]
ERMS and ARMS are fairly easy to distinguish based on
histology results but have overlapping cytological features.
90 Journal of Cytology / July 2008 / Volume 25 / Issue 3
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NB Synovial sarcoma DSRCT Wilm’s tumor
- + + -
- + + +
- - - -
+ - + -
- - + -
- - - -
Clinically, the age and site of presentation are useful in their
distinction. ERMS is common in the 1st decade of life and
occurs in the head, neck, and urogenital regions. ARMS occur
predominantly in the soft tissues of the limbs and are seen
in the 2nd and 3rd decades of life.[14]
The tumor cells vary from small, undifferentiated, round
cells to large pleomorphic cells with abundant eosinophilic
cytoplasm. The nuclei are large with marked pleomorphism.
Aspiration smears from this tumor reveal cells that vary
considerably in size and shape, but usually contain moderate
to abundant amounts of cytoplasm which stain deep blue and
contain occasional, small, cytoplasmic vacuoles.[15] A variable
proportion of cells is much larger with abundant pale blue
to grey, opaque cytoplasm. Within the cytoplasm of some of
these cells is an ill-defined, relatively dense, inclusion-like
area indicating myogenic differentiation. Tumors with this
morphology are predominantly composed of undifferentiated
cells with only occasional cells showing features of myogenic
differentiation. However, precise distinction between these
subtypes of RMS may not be possible in all cases and many
require the application of special techniques.[16]
In the vast majority of cases, the immunocytochemistry
panel includes myogenic markers such as desmin, myoglobin,
muscle-specific actin, and sometimes, a more specific marker
(MyoD1).[17,18]
ARMS is characterized by translocation involving the FKHR
gene on chr-13 and Pax3/7 gene on chromosome 2 or 1
respectively. Among these tumors, 90% show t(2:13) q
(35:Q14) and 10% show variant t(1:13) (p36q14). Both PAX3
and PAX7 regions encode related DNA binding domains
which may alter the expression of a common group of target
genes.[19]
Wilm’s tumor: Wilm’s tumor (WT) is a common pediatric,
malignant tumor. It is seen primarily in infants and 50% of the
cases occur before the age of three years. It is a malignant
neoplasm of the kidney which morphologically resembles
embryonal renal tissue. FNAC of WT reveals blastemal tissue
characterized by loosely arranged, small, undifferentiated
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Rajwanshi: Cytology of round cell tumors
cells in which there are scattered large, pale-staining cells
disposed in a random fashion. Tubular differentiation is
represented by a circular arrangement of larger, pale-staining,
epithelial cells with smaller, more undifferentiated cells at the
periphery.[20] Mesenchymal elements are usually composed of
spindle-shaped cells, although occasional, large pleomorphic
cells with abundant cytoplasm may be seen. Although the
diagnosis can be readily made when all three components
(blastemal, epithelial, and mesenchymal) of WT are present,
some tumors only show the blastemal component in which
case, ancillary studies such as electron microscopy and
ICC play an important role in making a correct diagnosis.
Positivity for CK, EMA, and VIM is seen in the majority of
cases whereas NSE positivity can be a pitfall in the diagnosis
of WT.[21]
Synovial sarcoma: Synovial sarcomas which account
for 5–10% of soft tissue sarcomas, typically arise in the
paraarticular region in adolescents and young adults. Two
major histological subtypes are the classic biphasic and the
monophasic types. The biphasic type contains both epithelial
and spindle cells,[22] whereas the monophasic type is entirely
composed of spindle cells. Accurate categorization usually
requires ICC and other ancillary studies. EMA positivity is seen
in 95% of the cases whereas CK positivity is seen in 40% of the
cases.[23] Cytogenetic studies have revealed a characteristic
chromosomal translocation L(x18,) in more than 90% of the
tumors. Cloning of the translocation breakpoints showed that
L(x:18) results in the fusion of two novel genes, designated
syt at (18q11) and ssx(xpn). The xp11 breakpoint involves two
closely related genes, ssx1 and ssx2 located in the vicinity of
the ornithine aminotransferase-like (OATL) pseudogenes 1 and 2
respectively. The L(x:18) results in the formation of a chimeric
protein which can be detected by RT-PCR.[24]
Desmoplastic small round cell tumor: This tumor typically
occurs in adolescent males and may be located in the
abdomen, pelvis, retroperitoneum, scrotum, or pleura. It
shows a nesting growth pattern with an intense desmoplastic
stroma and focal epithelial differentiation in some areas.[1]
Aspiration smears are characterized by undifferentiated
round cells arranged in sheets and sometimes surrounded
by dense, collagenous stroma. When the desmoplastic
stroma is not represented, a morphological diagnosis may
be difficult.[25] Characteristically, IHC reveals staining for
CK and desmin.[26] Molecular studies have shown a specific
reciprocal translocation, t(11:22) (p13q12). The molecular
characterization of the breakpoint rejoin has revealed the
creation of a fusion gene between the EWS gene and Wilm’s
tumor gene (WT). There is a fusion of the EWS gene on
Journal of Cytology / July 2008 / Volume 25 / Issue 3
chromosome 22 with WTI gene on chromosome 11, which
can be detected by RT-PCR.[27]
Malignant lymphoma: Small, noncleaved cell lymphomas and
lymphoblastic lymphomas constitute the great majority of
childhood lymphomas. Although rare, large cell lymphomas
may be seen in children. Although the ICC of FNA material is
usually adequate for establishing the diagnosis of malignant
lymphomas, the precise phenotype may best be determined
by FCM which usually provides adequate quantization of
antigen expression. Immunoreactivity of the neoplastic cells
for LCA (CD45) defines the tumor as a lymphoma.[1]
In cases of Burkitt’s and nonBurkitt’s lymphomas, demonstration
of the characteristic chromosomal translocation t(8:14)
involving the c-myc protooncogene on chromosome 8 and the
gene for the heavy chain of immunoglobulin on chromosome
14 may help confirm the diagnosis. In some cases, there
may be a t(2:8) chromosomal translocation involving c-myc
and kappa light chain genes and t(8:22) involving c-myc and
the lambda light chain genes. These translocations can be
readily recognized by routine cytogenetics or by using F1SH
or RT-PCR.
References
1. Akhtar M, Iqbal MA, Mourad W, Ali MA. Fine-needle aspiration biopsy
diagnosis of small round cell tumors of childhood: A comprehensive
approach. Diagn Cytopathol 1999;21:81-91.
2. Layfield LJ, Glasgow B, Ostrzega N, Reynolds CP. Fine-needle aspira-
tion cytology and the diagnosis of neoplasms in the pediatric age group.
Diagn Cytopathol 1991;7:451-61.
3. Brahmi U, Rajwanshi A, Joshi K, Dey P, Vohra H, Ganguly NK, et al.
Flow cytometric immunophenotyping and comparison with immuno-
cytochemistry in small round cell tumors. Anal Quant Cytol Histol
2001;23:405-12.
4. Sahu K, Pai RR, Khadilkar UN. Fine needle aspiration cytology of the
Ewing’s sarcoma family of tumors. Acta Cytol 2000;44:332-6.
5. Kovar H, Dworzak M, Strehl S, Schnell E, Ambros IM, Ambros PF,
et al. Overexpression of the pseudoautosomal gene MIC2 in Ewing’s
sarcoma and peripheral primitive neuroectodermal tumor. Oncogene
1990;5:1067-70.
6. Ramani P, Rampling D, Link M. Immunocytochemical study of 12E7 in
small round-cell tumors of childhood: An assessment of its sensitivity
and specificity. Histopathology 1993;23:557-61.
7. Riopel M, Dickman PS, Link MP, Perlman EJ. MIC2 analysis in pediatric
lymphomas and leukemias. Hum Pathol 1994;25:396-9.
8. Ambros IM, Ambros PF, Strehl S, Kovar H, Gadner H, Kuntschik MS.
MIC2 is a specific marker for Ewing’s sarcoma and peripheral primi-
tive neuroectodermal tumors: Evidence for a common histogenesis of
Ewing’s sarcoma and peripheral primitive neuroectodermal tumors
from MIC2 expression and specific chromosome aberration. Cancer
1991;67:1886-93.
9. Rossi S, Orvieto E, Furlanetto A, Laurino L, Ninfo V, Dei Tos AP.
Utility of the immunohistochemical detection of FLI-1 expression in
round cell and vascular neoplasm using a monoclonal antibody. Mod
Pathol 2004;17:547-52.
91
CMYK91
[Downloaded free from http://www.jcytol.org on Thursday, April 5, 2018, IP: 179.184.129.82]
Rajwanshi: Cytology of round cell tumors
10. de Alava E, Gerald WL. Molecular biology of the Ewing’s sarcoma/prim-
itive neuroectodermal tumor family. J Clin Oncol 2000;18:204-13.
11. Miettinen M, Chattern J, Paetau A, Stevenson A. Monoclonal antibody
NB84 in the differential diagnosis of neuroblastoma and other small
round cell tumors. Am J Surg Pathol 1998;22:327-32.
12. Caillaud JM, Martinez-Madrigal F, Hartmann O, Carlu C. Immunohis-
tochemical demonstration of neurone specific enolase in bone marrow
infiltrated by neuroblastoma. J Clin Pathol 1991;44:309-12.
13. Burchill SA, Bradbury FM, Smith B, Lewis IJ, Selby P. Neuroblastoma
cell detection by reverse transcriptase-polymerase chain reaction (RT-
PCR) for tyrosine hydroxylase mRNA. Int J Cancer 1994;57:671-5.
14. Folpe AL, McKenney JK, Bridge JA, Weiss SW. Sclerosing rhab-
domyosarcoma in adults: Report of four cases of a hyalinizing,
matrix-rich variant of rhabdomyosarcoma that may be confused with
osteosarcoma, chondrosarcoma, or angiosarcoma. Am J Surg Pathol
2002;26:1175-83.
15. Rajwanshi A, Rao KL, Marwaha RK, Nijhawan VS, Gupta SK. Role
of fine-needle aspiration cytology in childhood malignancies. Diagn
Cytopathol 1989;5:378-82.
16. Das DK. Fine-needle aspiration (FNA) cytology diagnosis of small
round cell tumors: Value and limitations. Indian J Pathol Microbiol
2004;47:309-18.
17. Rangdaeng S, Truong LD. Comparative Immunohistochemical staining
for desmin and muscle specific actin. A study of 576 cases. Am J Clin
Pathol 1991;96:32-45.
18. Dias P, Parham DM, Shapiro DN, Webber BL, Houghton PJ. Myogenic
regulatory protein (MyoD1) expression in childhood solid tumors: Diag-
nostic utility in rhabdomyosarcoma. Am J Pathol 1990;137:1283-91.
19. Shapiro DN, Sublett JE, Li B, Downing JR, Naeve CW. Fusion of PAX3
92 Journal of Cytology / July 2008 / Volume 25 / Issue 3
92CMYK
to a member of the forkhead family of transcription factors in human
alveolar rhabdomyosarcoma. Cancer Res 1993;53:5108-12.
20. Dey P, Radhika S, Rajwanshi A, Rao KL, Khajuria A, Nijhawan R, et al.
Aspiration cytology of Wilms’ tumor. Acta Cytol 1993;37:477-82.
21. Ellison DA, Silverman JF, Strausbauch PH, Wakely PE, Holbrook CT,
Joshi VV. Role of immunocytochemistry, electron microscopy and DNA
analysis in fine-needle aspiration biopsy diagnosis of Wilms’ tumor.
Diagn Cytopathol 1996;14:101-7.
22. Ladanyi M. The emerging molecular genetics of sarcoma translocations.
Diagn Mol Pathol 1995;4:162-73.
23. Silverman JF, Landreneau RJ, Sturgis CD, Raab SS, Fox KR, Jasnosz
KM, et al. Small-cell variant of synovial sarcoma: Fine-needle aspiration
with ancillary features and potential diagnostic pitfalls Diagn Cytopathol
2000;23:118-23.
24. De Leeuw B, Balemans M, OIde Weghuis D, Geurts van Kessel A.
Identification of two alternative fusion genes, SYT-SSX1 and SYT-
SSX2, in t(X; 18)(p11.2; q11.2)-positive synovial sarcomas. Hum Mol
Genet 1995;4:1097-9.
25. Crapanzano JP, Cardillo M, Lin O, Zakowski MF. Cytology of desmo-
plastic small round cell tumor. Cancer 2002;96:21-31.
26. Dave B, Shet T, Chinoy R. Desmoplastic round cell tumor of childhood:
can cytology with immunocytochemistry serve as an alternative for tissue
diagnosis? Diagn Cytopathol 2005;32:330-5.
27. Sawyer JR, Tryka AF, Lewis JM. A novel reciprocal chromosome
translocation t(11;22)(p13;q12) in an intraabdominal desmoplastic small
round-cell tumor. Am J Surg Pathol 1992;16:411-6.
Source of Support: Nil, Conflict of Interest: None declared.