2015, Volume 31, Number 1, Page(s) 016-023
Diagnostic Power and Pitfalls of Intraoperative Consultation (Frozen Section) in Rhabdomyosarcoma
Olcay KURTULAN, Kemal KÖSEMEHMETOĞLU
Department of Pathology, Hacettepe University, Faculty of Medicine, ANKARA, TURKEY
Keywords: Rhabdomyosarcoma, Sarcoma, Cytology, Diagnostic errors
Intraoperative consultation plays an important role in the
management of soft tissue sarcomas, such as rhabdomyosarcoma. In
this study, we aimed to draw attention to the important points during
frozen section interpretation, and analyse the accuracy of frozen
diagnosis in rhabdomyosarcoma patients.
Material and Method: The cases, both diagnosed as
rhabdomyosarcoma or followed with a history of rhabdomyosarcoma,
and interpreted with intraoperative consultation (frozen section)
between 2000 and 2013 were culled from pathology archives. The
diagnoses were confirmed by desmin and myogenin, immunohistochemically.
The frozen and final diagnoses were noted of 21 biopsy
specimens of 19 patients. Sensitivity and specificity of intraoperative
consultation were calculated regarding to the major diagnostic
discrepancies leading to a change in surgical management of the
patient, after exclusion of the cases deferred to paraffin section.
Results: Of the evaluated 21 biopsy material, 3 (14%) were
misdiagnosed: Of the 2 false negative embryonal rhabdomyosarcoma
cases, sample was not representative of the tumor, and there
was chemo/radiotherapy induced changes in the other case. In
the only false positively diagnosed case with a known history of
rhabdomyosarcoma, inflammatory cells were misinterpreted as small
round cell neoplasm. In 5 (29%) of 21 biopsies, a frozen diagnosis
could not be given, and the diagnosis was deferred. Six cases (29%)
were evaluated with cytological squash or imprint preparation;
none of the misdiagnosed cases was evaluated with adjunct
cytological preparation. Six of 8 misdiagnosed or deferred biopsies
showed morphological changes secondary to radiotherapy and/or
chemotherapy. Sensitivity, specificity, positive predictive value, and
negative predictive value were calculated as 85%, 67%, 92% and 50%,
Conclusion: Intraoperative consultation for rhabdomyosarcoma is
a reliable tool with high sensitivity and fair specificity. Cases with
treatment effect may lead to diagnostic difficulties, especially false
negative results. Understanding the diagnostic algorithm of surgeon
may prevent misdiagnosis of frozen specimen. Our results also
emphasize the diagnostic role of intraoperative cytology as an adjunct
to frozen section.
Surgical pathologists inevitably have to deal with soft tissue
tumours not only on routine pathology workup, but also
at frozen section. Nevertheless, there are encouraging
results in the literature designating the reliability of frozen
section in soft tissue tumors for general rather than exact
diagnosis in defining the previously unknown pathologic
. Intraoperative consultation may have some
important role, especially for determination of surgical
margins, presence of diagnostic material, and separation of
fresh material for molecular studies in the management of
rhabdomyosarcoma (RMS), the most common sarcoma of
children and adolescents2,3
In this study, we assessed the diagnostic accuracy of
intraoperative consultation in RMS in our department. We
aimed to emphasize the reasons leading to misdiagnosis,
and to define the specific morphologic findings to reach the
We re-examined the slides of 25 biopsies from 23 patients
that underwent frozen-section evaluation for RMS between
the years 2000 and 2013. Immunohistochemical studies for
desmin (Biocare, Concord/CA, 1/50) and myogenin (DBS,
Pleasenton/CA, 1/50) were performed using Leica Bond
Polymer Refine Detection Kit (Leica, DS9800) and Leica
Bond Max Autostainer Machine to confirm the diagnoses,
and the presence of “PAX3-FOXO1A” fusion was shown
in 3 cases of alveolar RMS. After review, 4 cases whose
diagnoses could not be confirmed were excluded from
the study. Remaining 21 biopsies from 19 patients were
included in the study, and morphological features were
Only the major differences that change the surgical
management were considered for the assessment of
diagnostic power (sensitivity, specificity, and predictive
values) of frozen-section diagnoses.
Eight cases were male and 11 were female. The mean age
was 16.5 (ranged between 1 and 68), and 12 (63%) were
under the age of 15. After the re-examination of the slides,
11 cases (52%) were diagnosed as embryonal RMS, 5 cases
(24%) as alveolar RMS, and 1 case (5%) as pleomorphic
RMS. Remaining 4 (19%) cases were negative for tumour; 3
were followed-up with the previous diagnosis of embryonal
RMS and 1 patient was only suspected for RMS. The tumour
was localized to the head and neck in 15 patients (79%), and
the uterus, paravertebral region and prostate in 1 for each
(5%). Localization of tumour in 1 case was not available.
Three of the 21 biopsies (14%) were misdiagnosed in
intraoperative consultation; 2 had false negative, and 1 had
false positive diagnoses (Table I). In one of the false negative
cases, tumour was not represented in the small frozen biopsy
material sent by surgeon (Figure 1A). Complete examination
of the biopsy material sent to pathology department revealed
hypercellular areas composed of neoplastic cells having round,
monomorphic, hyperchromatic nuclei, and scanty cytoplasm
(Figure 1B) besides the hypocellular and hyalinized zones
which were seen in frozen section. Immunohistochemically,
desmin and myogenin were positive in the neoplastic cells
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|Figure 1: A) Hyalinized, hypocellular areas devoid of tumour on frozen section (H&E; x200). B) Presence of hypercellular neoplasia
on the right side and hyalinized areas on the left representing the areas seen in frozen section on paraffin section (H&E; x100).
C) Cytoplasmic expression of desmin in the neoplastic cells (Desmin; x100). D) Nuclear myogenin expression (Myogenin; x200).
In the other false negative case, the biopsy showed
prominent chemo/radiotherapy related changes (Figure
2A-D), characterized by oedema and fibrosis with
inflammatory background, and only a few atypical cells were present (Figure 2A). In the permanent section,
atypical cells were present in the background of fibrosis
and inflammation (Figure 2B). Atypical cells were in the
form of strap cells with unipolar or bipolar eosinophilic
cytoplasmic extensions, and hyperchromatic, irregular
nuclei. Histological hallmarks, such as broken arrow sign (Figure 2C), and immunohistochemical demonstration of
myoid markers desmin and myogenin were diagnostic for
RMS (Figure 2C,D).
Only one false positive case, previously diagnosed as
RMS, and re-excised because of surgical margin positivity,
was evaluated as having small blue round cell tumour in frozen section (Figure 3A,B); however, it turned out to
be an intensive inflammatory infiltrate and fibrosis in the
permanent section (Figure 3C), and immunohistochemical
analysis for myogenin was negative (Figure 3D).
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|Figure 2: A) Oedematous and fibrotic changes on frozen section (H&E; x100). B) Paraffin section of frozen material; atypical cells are
seen in the background of fibrosis and inflammation (H&E; x200). Some cells also show diagnostic striations (inset; x400) C) Elongated
cytoplasmic expression of desmin in the neoplastic cells corresponding to the broken arrow sign in H&E (inset) (H&E; x 400). D) Nuclear
expression of myogenin (Myogenin; x400).
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|Figure 3: A,B) Frozen section: Intensive infiltrate of lymphocytes on vessel-rich background misinterpreted as small blue round cell
tumour. C) Paraffin embedded section; Presence of chronic inflammation, granulation tissue and fibrosis was easier to recognize
(H&E; x200) D) Myogenin was also negative (Myogenin; x200).
Five biopsies (24%) were reported as indeterminate during
intraoperative consultation, and definitive diagnosis was left
to permanent paraffin sections (Table I). Six out of 8 patients
(75%) who were misdiagnosed or had inconclusive result during frozen section previously received chemotherapy
or radiotherapy, and biopsies of these patients showed
significant treatment related morphological changes,
such as fibrosis, hyalinization, and inflammatory reaction
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|Figure 4: A,B) Fibroblast-like cells within collagenous background and hemosiderin pigment in both frozen and permanent sections.
Due to the presence of scattered hyperchromatic cells, frozen diagnosis was “suspicious for malignancy, definitive diagnosis should
be given in permanent sections” (H&E; x100 and x200). C) Examination of remaining surgical material in paraffin sections revealed
rhabdomyoblastic strap cells with unipolar or bipolar eosinophilic cytoplasmic extensions and hyperchromatic nuclei (H&E; x200).
D) Positive myogenin staining (circles) supported the diagnosis of RMS (Myogenin; x200).
Sensitivity, specificity, positive and negative predictive
values of intraoperative consultation diagnosis for RMS
were calculated as 85%, 67%, 92%, and 50%, respectively.
Six of 21 cases (29%) were evaluated with cytological
preparations, and none of them was misdiagnosed in
the intraoperative consultation. Also, squash smear
examination was not performed in none of the cases with false frozen diagnoses. Microscopically cytological
preparations were hypercellular, and had neoplastic cells
with small, round, hyperchromatic nuclei, and scanty
cytoplasm (Figure 5A,B).
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|Figure 5: Cytological features of RMS in squash smears during intraoperative consultation (H&E; x200 and x400).
Rhabdomyosarcoma (RMS) is a primitive, malignant softtissue
tumour with phenotypical and biological features
of embryonic skeletal-muscle cells. RMS has 4 distinct
subtypes: embryonal, alveolar, pleomorphic, and spindle
cell/sclerosing RMS, each having diverse morphology and
. What makes RMS the subject of
the current study is that RMS is the most common softtissue
sarcoma in children and adolescents, thus more
frequently encountered in frozen section than any other
rarer sarcomas. Moreover, in the USA, frozen triage of
childhood RMS is highly recommended for national
which is also expected to be valid for
Turkey in the near future.
In soft tissue tumours, intraoperative consultation is
useful in determining the surgical procedure, in assessing
if the tumour is present and in constructing a differential
diagnosis that can direct the proper triage of tissue for flow
cytometry, electron microscopy, and molecular studies/
cytogenetics, particularly for neuroblastoma and RMS2.
Tissue triage is optimally performed at the time of frozen
section. In many cases, it is important that a portion of
tissue be submitted for ancillary studies, even from fineneedle
aspiration (FNA) and core needle biopsy specimens,
after sufficient tissue has been submitted for histological
Frozen diagnosis for RMS is found to be highly sensitive,
reaching 85%; however, specificity is lacking, slightly better
than flipping a coin. Therefore, it’s recommended to be
more careful while one decides to give “negative” result
in frozen sections because of low specificity and negative
predictive value. One should be especially careful about
morphological pathognomonic clues, such as broken
arrow sign and cross-striations, and a cytological squash
preparation should be used as an auxiliary technique in
order to prevent false negative results. Also, a complete
understanding of the surgeon’s treatment algorithm is
recommended before rendering a frozen section diagnosis6. According to the algorithm determination, the case
could be released to paraffin embedded sections.
In our series, treatment-related morphological changes
seem to have an important role leading to a false frozen
section diagnosis. It is important to get the clinical
history of patient if he/she had a working diagnosis of
RMS previously or had received treatment. A variety of
therapy-related histological changes ranging from necrosis,
chronic inflammation, fibrosis to atrophic or regenerating
nonneoplastic skeletal muscle have been reported in
most RMSs, especially the embryonal subtype7. Posttreatment
specimens can show fibrosis, inflammation with
macrophages and lymphocytes, and scattered neoplastic
cells simulating reactive fibroblasts. Interpretation of
therapy-induced differentiations is a great challenge for the pathologists and it is quite difficult to evaluate them
in the frozen sections. The use of cytological preparations
as an adjunct to frozen section is highly reliable tool8,
and may be quite useful for the differentiation of chronic
inflammatory cells from small round rhabdomyoblasts
and for the straightaway appreciation of the morphological
details of the neoplastic cells.
Finally, we conclude that intraoperative consultation for
RMS is a quite reliable tool with high sensitivity and fair
specificity. Pathologists dealing with frozen section should
be aware of treatment effect that may lead to diagnostic
difficulties, especially false negative results. Understanding
the diagnostic algorithm of the surgeon is highly
recommended to prevent misdiagnosis. Our findings also
encourage the use of intraoperative cytology as an adjunct
to frozen section.
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