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Ultrastructural Pathology, iFirst:1–5, 2010
Copyright & Informa Healthcare USA, Inc.
ISSN: 0191-3123 print/1521-0758 online
DOI: 10.3109/01913123.2010.517899
Abnormal Nuclear Structures (Micronuclei,
Nuclear Blebs, Strings, and Pockets) in a
Case of Anaplastic Giant Cell Carcinoma of
the Thyroid: An Immunohistochemical and
Ultrastructural Study
Rosario Alberto Caruso, MD
Francesco Fedele, MD, and
Costantino Crisafulli, PhD
Department of Human Pathology,
University Hospital, Messina, Italy
Domenica Paparo, MD and
Antonino Parisi, MD
Roberta Lucianò, MD and
Vittorio Cavallari, MD
Department of Surgical Sciences,
University Hospital, Messina,
Italy
Department of Human Pathology,
University Hospital, Messina, Italy
ABSTRACT
The authors report a case of a 70-year-old woman with an anaplastic giant cell thyroid carcinoma,
along with immunohistochemical and electron microscopic findings. Histologically, the tumor is
characterized by mononucleated and multinucleated giant cells, lack of architectural cohesion,
atypical mitoses, and extensive areas of coagulative necrosis. Tumor cells showed AE1/AE3
positivity as well as nuclear overexpression of p53 and ki-67. Semithin sections revealed multiple
nuclei with heterogeneous size ranging from micronuclei to large-size (giant) nuclei. Micronuclei
were confirmed by electron microscopy that disclosed also the presence of nuclear blebs, strings,
and pockets. Morphological findings of these abnormal nuclear structures in conjunction with p53
and Ki-67 nuclear overexpression suggested a faulty mitotic checkpoint/mitotic catastrophe in the
progression of anaplastic giant cell thyroid carcinoma.
Keywords: abnormal nuclear structures, anaplastic giant cell-type, micronuclei, thyroid carcinoma
Anaplastic thyroid carcinomas, which account for less
than 2% of all thyroid cancers, are among the most
aggressive human malignancies and are nearly always
fatal [13]. Microscopically, three histological patterns
are commonly described: spindle, giant cell, and
squamoid [1,2,14]. It is believed that anaplastic thyroid
carcinoma is derived from follicular epithelial cells and
represents a terminal dedifferentiation of preexisting
differentiated carcinoma [12].
In an effort to bring additional information to the
understanding of the morphologic characteristics of
this rare tumor, we investigated light and electron
microscopic characteristics of the nuclei in a case of
anaplastic giant cell thyroid carcinoma associated with
foci of differentiated carcinoma. These studies reveal
previously unreported abnormal nuclear structures,
including micronuclei as well as nuclear blebs, strings,
and pockets, which were associated with an increased
Received 2 August 2010; accepted 3 August 2010.
Address correspondence to Prof. Rosario A. Caruso, Dipartimento di Patologia Umana, Policlinico Universitario, I-98125 Messina, Italy. E-mail:
[email protected]
1
R. A. Caruso et al.
frequency of abnormal mitoses as assessed by Ki-67
immunolabeled paraffin sections of the same tumor.
Moreover, a p53 immunohistochemical overexpression
was also documented. The implications of nuclear
changes in the dedifferentiation progression of the
tumor reported here are discussed.
CLINICAL HISTORY
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A 70-year-old woman presented with a growing,
painless anterior neck mass that had existed for 2
months and was associated with dysphagia and hoarseness. There was no family history of thyroid disease and
the patient had no history of radiation exposure to the
neck region. Levels of serum-free thyroid hormones,
thyreotropin, and calcitonin were within the normal
range. A needle aspirate of the mass demonstrated a
noncohesive population of tumor cells with pleomorphic, vesicular nuclei containing prominent nucleoli
and abundant cytoplasm. The smears were blood-mixed
but necrosis or inflammation was not a conspicuous
feature. Total thyroidectomy was performed. The patient
rapidly developed local recurrence with massive mediastinal lymph node metastases, and died 2 months after
surgery. No autopsy was performed.
cut surface showed a multinodular aspect with nodule,
maximum of 2.5 cm in diameter. Microscopic findings
showed the tumor consisted of a solid sheet of poorly
cohesive mononucleated and multinucleated giant cells
(Figure 1). The stroma was scanty, and there were focal
areas of coagulative necrosis. H & E staining showed
foci of papillary tumor, organized in irregular follicles,
lined by cells with clear, overlapping nuclei (Figure 1).
Atypical mitotic figures were frequent, reaching 20
mitoses per 10 high-power fields. Araldite-embedded
semithin sections showed multiple micronuclei in giant
multinucleated cells (Figure 2). Micronuclei were round
or oval in shape with a diameter not exceeding onethird of the diameter of the main nucleus. They had the
MATERIALS AND METHODS
For light microscopy, the specimens were fixed in 10%
formalin for 24 h at room temperature and embedded in
paraffin. Sections were stained with hematoxylin–eosin
(H & E). Immunohistochemistry was performed using
paraffin-embedded material and the avidin biotin
immunoperoxidase technique. Primary antibodies used
in immunohistochemistry were thyroglobulin (DAK-Tg6,
dilution 1/200), EMA (E29, dilution 1/200), cytokeratin
(AE1/AE3, dilution 1/50), calcitonin (CAL-3-F5, dilution
1/50), desmin (D33, dilution 1/100), smooth muscle actin
(1A4, dilution 1/50), CD68 (KP1, dilution 1/50), Ki-67
(MIB-1, dilution 1/50), and p53 (DO-7, dilution 1/50,
all from DakoCytomation (Glostrup, Denmark). As
controls, known positive tissue sections and negative
controls devoid of primary antibody were used. For
electron microscopy, fresh specimens were fixed in 3%
phosphate-buffered glutaraldeyde, pH 7.4, and postfixed
in 1% osmium tetroxide. Semi-thin araldite-embedded
sections were stained with toluidine blue for selection of
fields. Thin sections were double-stained with uranyl
acetate and lead citrate; they were then examined and
photographed with a Zeiss EM 902 electron microscope
(Carl Zeiss, Oberkochen, Germany).
RESULTS
2
Macroscopic findings revealed that the thyroid had a
lobular appearance and measured 7 3 6 3 5 cm. The
Figure 1. Follicular variant of papillary carcinoma, showing
clear, overlapping nuclei, is observed adjacent to anaplastic
tumor. H & E, 3100.
Figure 2. Large patches of giant tumor cells containing multiple nuclei with a vesicular chromatin pattern and one or
more large nucleoli. The large giant tumor cell shows several
micronuclei (arrows). Semithin section; toluidine blue 3400.
Anaplastic Giant Cell Carcinoma of the Thyroid
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same staining intensity and texture as the main nucleus
and were distinctly observed within the cytoplasm of
tumor cells (Figure 2).
Immunohistochemical results for epithelial markers
(cytokeratin AE1/AE3 and EMA) showed only a few
scattered, weakly positive cells. Cytokeratins and
thyreoglobulin stained in the more differentiated
papillary tumor cells. Immunostaining for desmin,
smooth muscle actin, thyroglobulin, and CD68 were
negative in tumor cells, but stained normal components
of surrounding tissues. The Ki-67 positive rate was 94%
of the mononuclear cells and 85% of the multinucleated
giant cells, whereas in the well-differentiated cell
component was 8%. Furthermore, aberrant mitotic
figures were decorated by Ki-67 immunostaining in
multinucleated giant tumor cells (Figure 3). Intense
nuclear staining of p53 was found in the anaplastic
areas, but no positive cells were observed in either
neighboring papillary thyroid tumor cells (Figure 4) or
normal follicular thyroid epithelial cells (not shown).
Electron microscopy disclosed several abnormal
nuclear structures, including nuclear strings and
micronuclei, as well as nuclear blebs and pockets.
Micronuclei were surrounded by a double membrane
and contained a mixture of euchromatin and heterochromatin (Figure 5). Figure 6 shows a nuclear string
connecting two lobes of a nucleus. Nuclear pockets are
also present and are formed by apposed extensions of
nuclear membranes, including their fibrous lamina
and subjacent chromatin. Nuclear blebs are morphologically similar to micronuclei with the exception that
they are joined to the nucleus by a thin nucleoplasmic
connection (Figure 6). Furthermore, electron microscopy revealed a variable presence of large vesicular
mitochondria and few microvilli, whereas endocrine
Figure 3. Atypical mitoses of giant tumor cell. Note the
nuclei with Ki-671 chromosomes, resembling prophase. 3400.
granules were not found. The cells were joined by a
few rudimentary cell junctions with broad intercellular
spaces into which interdigitating, microvilli-like cell
processes project partially. A basement membrane was
absent (Figure 5).
Figure 4. Strong p53 staining is seen in the nuclei of the
anaplastic tumor cells but lack of p53 immunoreactivity is
seen in the neighboring papillary thyroid tumor cells. 3200.
Figure 5. Anaplastic carcinoma cell showing multiple
nuclei, heterogeneous in size, and micronuclei (arrowhead).
Nuclear projections (thick arrow) and blebs (thin arrow)
are also present. Tumor cells are joined by a few rudimentary cell junctions. The cytoplasm of tumor cells contains a
variable quantity of large vesicular mitochondria and a few
microvilli. 38000.
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R. A. Caruso et al.
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string was defined as a chromatin thread connected to
the membrane(s) of one or two nuclei, a nuclear bleb as
a round or oval protrusion of the nuclear membrane
connected to the main part of the nucleus by a thinner
chromatin segment, and a micronucleus as a rounded
chromatin located adjacent to a nucleus, with a
diameter not exceeding one-third of the diameter of
that nucleus. Nuclear heterogeneity has been documented by Fenech et al. [5] in normal and folatedeprived cultures of human lymphocytes. In addition
to micronuclei, they describe nuclear buds and
nucleoplasmic bridges, which are synonymous with
the nuclear blebs and strings of Gisselsson’s study [8],
respectively. Thus, abnormal nuclear shape in solid
tumors is an indicator of mitotic dysregulation, which
may prompt further studies of mitotic instability [9].
Figure 6. Nuclear string connecting two lobes of a nucleus
(arrowhead). Nuclear membranes arch over to rejoin the
main nuclear mass, enclosing a portion of cytoplasm and
forming a nuclear pocket (double arrow). Note a nuclear
bleb containing micronucleus (arrow). 38000.
DISCUSSION
We present a case of anaplastic thyroid carcinoma that
coexisted with a follicular variant of papillary carcinoma. The preponderant pattern in anaplastic carcinoma was the pleomorphic giant cell that may be
confused with sarcoma. In our case, focal immunoreactivity for cytokeratin AE1/AE3 and ultrastructural
evidence of microvilli, often forming complex interdigitations, and rudimentary desmosomes, favour
the diagnosis of an epithelial neoplasm. Medullary
thyroid carcinoma rarely undergoes dedifferentiation/
anaplastic transformation and loses immunoreactivity
for calcitonin and CEA. Neurosecretory granules were
not seen at the ultrastructural level, effectively excluding this consideration [1]. Anaplastic carcinoma with
osteoclast-like giant cells is a rare variant and should
also be considered in the differential diagnosis.
Osteoclast-type giant cells are immunoreactive for
CD68, but not for cytokeratin. They contain bland
nuclei, which are different from the surrounding
highly pleomorphic nuclei of carcinoma cells [6].
4
The nuclei of the cells of most solid tumors in
histopathologic preparations vary in size, shape, and
chromatin pattern, both from normal nuclei and from
each other. Nevertheless, only a few attempts have
been made to classify abnormalities in the morphology
of interphase nuclei. The study of Gisselsson et al. [8],
on primary cultures of solid tumors, suggested a
rough classification into three types: nuclear strings,
nuclear blebs, and micronuclei. In that study, a nuclear
Our case of pleomorphic giant cell carcinoma of the
thyroid is characterized not only by abnormally large
giant nuclei, but also by those that are abnormally small,
in the form of micronuclei. In our study, araldite
semithin sections permitted more precise evaluation of
micronuclei than paraffin sections. Electron microscopy
not only confirmed the presence of micronuclei, but
also disclosed nuclear blebs, pockets, and strings, all
morphological features suggesting the occurrence of
an ‘‘amitotic nuclear division’’. A Ki-67 immunohistochemical analysis revealed a mitotic ability of the
multinucleated giant cells, thus confirming the occurrence of acytokinetic multinucleation.
Tissue necrosis is absent in differentiated thyroid
carcinomas, while it is a common finding in poorly
differentiated and anaplastic thyroid carcinomas [10].
Along the same lines, gene p53 mutations are
restricted to aggressive tumors (17–38% of poorly
differentiated thyroid carcinomas and 67–88% of
undifferentiated thyroid carcinomas as opposed to
0–9% of well-differentiated thyroid carcinomas)
[3,4,11]. In the anaplastic areas of our thyroid
neoplasm a strong overexpression of p53 protein was
detected with the DO-7 antibody, confirming previous
data in literature.
Micronuclei and multinucleation (the presence of 2 or
more nuclei with similar or heterogeneous size,
resulting from deficient separation during cytokinesis)
as well as abnormal mitoses are morphologic aspects
of mitotic catastrophe, a common phenomenon occurring in tumor cells in vitro with impaired p53 function
exposed to various cytotoxic and genotoxic agents
[7,15]. Mitotic catastrophe often results in the generation of aneuploid and polyploidy cell progeny, as also
reported in anaplastic thyroid carcinoma [12]. The
defective p53 checkpoint causes the improper segregation of chromosomes, resulting in aberrant mitosis,
multiple micronuclei, and an eventual necrosis-like
death [15]. In our case, necrosis occurs in an extensive
Anaplastic Giant Cell Carcinoma of the Thyroid
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background of Ki-67-positive cycling tumor cells.
Furthermore, Ki-67 immunohistochemistry decorated
abnormal multipolar mitoses. Taken together, high
mitotic activity, necrosis, multinucleation, abnormal
nuclear structures (micronuclei, nuclear blebs, strings,
and pockets), and p53 and Ki-67 overexpression
suggest the hypothesis of a faulty mitotic checkpoint
(mitotic catastrophe) in our case of anaplastic giant cell
thyroid carcinoma.
In conclusion, our light and electron microscopy
findings expand the nuclear morphology of anaplastic
giant cell carcinoma of thyroid to include micronuclei,
nuclear blebs (buds), and strings (nucleoplasmic
bridges). These previously unreported abnormal
nuclear structures were associated with p53 and Ki67 nuclear overexpression. Therefore, our data suggest
a faulty mitotic checkpoint (mitotic catastrophe),
described mainly in experimental models in vitro
and in vivo, and may be considered as expression of
the dedifferentiation process in this unusual, but
aggressive histological type of thyroid carcinoma.
Declaration of interest: The authors report no conflicts
of interest. The authors alone are responsible for the
content and writing of the paper.
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