1. Cell Membrane and Cytoplasmic Staining for MIB-1
in Hyalinizing Trabecular Adenoma of the
Thyroid Gland
Mitsuyoshi Hirokawa, M.D., Ph.D., and J. Aidan Carney, M.D., Ph.D.
The monoclonal MIB-1 antibody reacts with the nuclei of cells
in the late G1, S, G2, and M phases of the cell cycle. Previ-
ously, we found two cases of hyalinizing trabecular adenoma
that showed cell membrane and cytoplasmic immunopositivity
for the antibody. The purpose of this investigation was to con-
firm this exceptional reactive pattern of MIB-1 in hyalinizing
trabecular adenoma. For the study, we collected 13 additional
hyalinizing trabecular adenomas and stained a total of 15 tu-
mors using MIB-1 antibody. Ten cases of papillary thyroid
carcinoma were studied similarly. All hyalinizing trabecular
adenomas showed strong positivity for the antibody in 90% or
more of the tumor cells, localized especially to the cell mem-
brane and also to the cytoplasm. There was no cell membrane
or cytoplasmic MIB-1 positivity among the 10 papillary carci-
nomas. Luminal border of normal extratumoral thyroid follicles
rarely showed faint immunopositivity. Our findings indicate
that strong cell membrane and cytoplasmic immunoreactivity
for MIB-1 is a characteristic of the hyalinizing trabecular ad-
enoma. Staining for MIB-1 will be useful in differentiating
hyalinizing trabecular adenoma from papillary carcinoma,
which shares a number of cytologic and histologic findings
with hyalinizing trabecular adenoma.
Key Words: Thyroid—Hyalinizing trabecular adenoma—
MIB-1—Ki67—Immunohistochemistry—Papillary carcinoma.
Am J Surg Pathol 24(4): 575–578, 2000.
Hyalinizing trabecular adenoma of the thyroid gland is
a subtype of follicular adenoma described by Carney et
al.1
in 1987. This benign tumor shares several histologic
features with papillary carcinoma, including intranuclear
cytoplasmic invaginations, nuclear grooves, and psam-
moma bodylike formations. It coexists occasionally with
papillary carcinoma.1,8,10
These and other findings
have led to the suggestion that hyalinizing trabecular
adenoma and papillary carcinoma are closely related
tumors.2
In 1995, we reported two cases of hyalinizing trabec-
ular adenoma in which the cytoplasm of the tumor cells
was reactive for MIB-1.3
Because MIB-1 reacts with cell
nuclei in the late G1, S, G2, and M phases of the cell
cycle, the cytoplasmic immunoreactivity of MIB-1 in
hyalinizing trabecular adenoma was unexpected and un-
explained. We have now examined 13 additional cases of
hyalinizing trabecular adenoma for MIB-1 immunoreac-
tivity and found the same intense cytoplasmic positivity
for the antigen in all cases. Herein, we confirm the ex-
ceptional reactive pattern of MIB-1 in hyalinizing tra-
becular adenoma and point out its diagnostic usefulness
in differentiating the tumor from papillary carcinoma.
MATERIALS AND METHODS
We collected 15 cases of hyalinizing trabecular ade-
noma from the surgical pathology files of Kawasaki
Medical School, the University of Tokushima School of
Medicine, and the consultation files of the authors. He-
matoxylin– and eosin-stained slides of the tumors were
reviewed, and the diagnosis of hyalinizing trabecular ad-
enoma was confirmed. The patients included 13 women
and two men whose age ranged from 25 to 72 years
(median, 46.2 years). The tumors ranged in diameter
from 1.0 to 4.0 cm. Juxtaposed hyalinizing trabecular
adenoma and papillary carcinoma were present in one
patient.
Routinely processed, formalin-fixed, paraffin-embedded
tumor blocks were sectioned at 3 to 5 mm. After depa-
raffinization and rehydration, the tissue sections were
placed in 10 mol per liter citrate buffer (pH 6.0) and
heated for three periods of 5 minutes each in a micro-
wave oven at 500 W. The immunohistochemical proce-
dure was based on the avidin–biotin–peroxidase method.
From the Department of Pathology (M.H.), University of Tokushima
School of Medicine, Japan; and the Department of Laboratory
Medicine and Pathology (J.A.C.), Mayo Clinic and Mayo Foundation,
Rochester, MN, U.S.A.
Address correspondence and reprint requests to Mitsuyoshi
Hirokawa, MD, PhD, Department of Pathology, University of
Tokushima School of Medicine, 3-18-15 Kuramoto-cho, Tokushima,
770-8503, Japan.
The American Journal of Surgical Pathology 24(4): 575–578, 2000 © 2000 Lippincott Williams & Wilkins, Inc., Philadelphia
575

2. Sections were stained with monoclonal antibody MIB-1
(1:50 dilution; Immunotech, Marseilles, France). Lymph
node germinal center centrocytic nuclei were used as
positive controls. Negative control sections were stained
in parallel, the specific antibody having been replaced
with phosphate-buffered saline. The nuclear MIB-1-
positive rate was calculated by counting the number of
stained and unstained nuclei in randomly selected fields,
using a light microscope equipped with a 100× objective
lens and a 10× ocular lens. The result was expressed as
positive (labeled) nuclei per 1,000 nuclei counted.
Ten cases of conventional papillary thyroid carcinoma
from the surgical pathology files of Kawasaki Medical
School were studied similarly. These patients included
eight women and two men whose age ranged from 32 to
64 years (median, 48.8 years).
RESULTS
Hyalinizing Trabecular Adenoma
The limiting membrane of almost all of the tumor cells
was uniformly and strongly positive for MIB-1 (Fig. 1).
The reactivity was particularly intense in cell membrane
facing intratrabecular spaces or lumina, and sometimes
was located in discrete, round, oval, and elongated zones.
The cytoplasm was also positive but to a lesser degree,
with its peripheral portion showing slightly stronger im-
munoreactivity than the central (perinuclear) zone. The
occasional intranuclear cytoplasmic invaginations were
also weakly positive for MIB-1. Bipolar staining concen-
trated at the luminal and basal aspects of the cytoplasm
was present in one case. Nuclear positivity was observed
in a few tumor cells in two cases; the positive rate was
not estimated because nuclear staining was overwhelmed
by the cytoplasmic positivity. Negative controls showed
no reactivity.
Papillary Carcinoma
None of the papillary carcinomas showed cell mem-
brane or cytoplasmic positivity for MIB-1. Occasional
nuclei were stained (positive rate range, 0.78–3.14%;
Fig. 2).
FIG. 1. Hyalinizing trabecular adenoma. Tumor cells ex-
hibited strong cell membrane and less strong cytoplasmic
staining for MIB-1.
FIG. 2. Papillary carcinoma and hyalinizing trabecular ad-
enoma. Papillary carcinoma (top) showed nuclear immu-
noreactivity for MIB-1, compared with cell membrane and
cytoplasmic staining in hyalinizing trabecular adenoma
(bottom).
M. HIROKAWA AND J. A. CARNEY576
Am J Surg Pathol, Vol. 24, No. 4, 2000

3. Non-neoplastic Thyroid Follicles
The non-neoplastic cells lining the follicles surround-
ing the tumors were generally negative for MIB-1. An
occasional luminal border revealed weak positivity (Fig.
3). Rare nuclei of follicular epithelial cells also revealed
positivity (positive rate range, 0–0.58%).
DISCUSSION
The monoclonal antibody MIB-1 (Ki67) reacts with
antigens associated with human nuclear cell prolifera-
tion. These antigens are expressed during the G1, S, G2,
and M phases of the cell cycle and are absent in the G0
phase. An epitope of Ki67 is destroyed by formalin fixa-
tion and routine processing so that it can be studied only
in frozen sections. In contrast, the MIB-1 epitope is fixa-
tion resistant, permitting its use with formalin-fixed tis-
sues in conjunction with an antigen retrieval procedure.
These antibodies have been used in numerous studies
designed to predict the biologic behavior of tu-
mors.6,7,9,11
During these investigations, the antibodies
have reacted invariably with nuclei. High positive rates
for nuclear staining have been associated generally with
aggressive tumor behavior.
In 1995, we reported two cases of hyalinizing trabec-
ular adenoma that had intense cytoplasmic immunoreac-
tivity for MIB-1.3
In the current study, undertaken to
investigate further this phenomenon in a larger number
of cases, we report the MIB-1 findings in 13 additional
cases of the tumor. The results revealed the same pattern
of cell membrane and cytoplasmic positivity among the
new cases as was observed in our original two cases,3
thus confirming our earlier results. A few intranuclear
cytoplasmic invaginations were also positive for MIB-1.
Kaleem and Davila4
studied Ki67 immunoreactivity in a
case of hyalinizing trabecular adenoma and found an
incidence of nuclear positivity of 0.6%. The second case
in their report does not appear to be a hyalinizing tra-
becular adenoma because the hyalinization depicted in
the case illustration is intertrabecular and stromal, and
not intratrabecular and cytoplasmic. Rare nuclear posi-
tivity was observed in only two of our 15 cases of hya-
linizing trabecular adenoma. We could not determine the
positive rate precisely because cytoplasmic positivity
overwhelmed the nuclear staining.
To the best of our knowledge, the MIB-1 cell mem-
brane and cytoplasmic staining pattern we describe in
hyalinizing trabecular adenoma has not been reported
in other thyroid (or nonthyroid) tumors. Nor has there
been reference to the patchy positive staining of the
luminal cell membrane in rare normal thyroid follicles
that we observed. Katoh et al.5
studied nuclear MIB-
1 reactivity in a large series of neoplastic and non-
neoplastic thyroid conditions. Their material included
adenomas, normo/macrofollicular (n ‫ס‬ 14), tubular (n
‫ס‬ 18), solid/trabecular (n ‫ס‬ 6), and oxyphilic (n ‫ס‬ 5),
and carcinomas, papillary (n ‫ס‬ 60) and follicular
(n ‫ס‬ 14). They made no mention of finding cell mem-
brane or cytoplasmic positivity in any of the tumors. It is
improbable that the cytoplasm of the hyalinizing trabec-
ular adenoma cells and that of normal thyroid follicles
contains an epitope identical to the nuclear proliferation
antigen. More likely, both cell types have a cytoplasmic
antigen that cross-reacts with the anti-MIB-1 antibody.
The MIB-1 staining results we obtained with hyalin-
izing trabecular adenoma contrasted markedly with those
of papillary carcinoma, in which cell membrane and cy-
toplasmic staining was not present. Approximately 1% of
papillary carcinoma nuclei were immunopositive. Our
findings thus provide another morphologic difference be-
tween hyalinizing trabecular adenoma and papillary car-
cinoma, casting further doubt on the validity of the sug-
gestion of Fonseca et al.2
that the hyalinizing trabecular
adenoma is an encapsulated variant of papillary carci-
noma. In practice, staining for MIB-1 should prove to be
useful diagnostically in distinguishing between the two
tumors. ᮀ
FIG. 3. Normal thyroid tissue. Luminal border of follicular
lining cells is weakly positive for MIB-1 (arrows).
THYROID TRABECULAR ADENOMA 577
Am J Surg Pathol, Vol. 24, No. 4, 2000

4. Acknowledgment
The authors thank Dr. T. Manabe for access to cases at the
Department of Pathology, Kawasaki Medical School, Japan.
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