Tenascin Expression in Cancer Cells and Stroma of Human ...injected into the nude mouse subcutis (22). Thus, the TN in the breast cancer stroma is considered to be originated from
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Vol. 1, 1035-1041. September 1995 Clinical Cancer Research 1035
Tenascin Expression in Cancer Cells and Stroma of Human Breast
Cancer and Its Prognostic Significance1
Akinori Ishihara, Toshimichi Yoshida,
Hisao Tamaki, and Teruyo Sakakura2
Departments of Pathology [A. I.] and Surgery [H. T.], MatsusakaChuo General Hospital, Matsusaka, Mie 515, and Department ofPathology, Mie University, School of Medicine, 2-174 Edobashi,Tsu, Mie 514 [A. I., T. Y., T. S.], Japan
ABSTRACT
Sections of formalin-fixed, paraffin-embedded tissues
from 210 human breast cancers were immunohistochemi-cally examined using the mAb against human tenascin (TN)
RCB1. Immunoreactive TN was detected in the breast can-cer stroma in 77 (36.7%) cases, whereas the remaining 133
(63.3%) were negative. Of the 77, 12 (5.7%) cases also
showed positive staining in the carcinoma cell cytoplasm.The positive cells were often observed in the margin of thecancer nests at the site adjacent to the stroma. According to
the staining pattern of TN, the breast cancer cases wereclassified into the three groups of cancer cell TN(+)/stromal
TN(+), cancer cehl(-)/stromal TN(+), and cancer celh(-)/stromal TN(-). Analysis of the relationship of these TN
patterns with various clinicopathological characteristics ofthe tumors and the patient outcome revealed that, in corn-
parison to the cancer cell(-)/strornal TN(-) group, the
cancer cell TN(+)/stromal TN(+) group exhibited increasedfrequency of lymph node metastasis and exceptionally poor
outcome, and the cancer cehl(-)/stromal TN(+) group alsoshowed more frequent metastasis and poorer outcome. Most
of the cancer cell TN(+)/stromal TN(+) cases were c-erbB-2
positive and estrogen receptor negative. Furthermore, in situ
hybridization of freshhy obtained breast cancer tissues dem-
onstrated that both cancer cells and stromal cells express TN
mRNA. These results indicate that the TN in breast cancer
is produced by cancer epithehial cells as well as by stromalmesenchymal cells, and that cancer cell TN might be in-
volved in cancer spreading, resulting in unfavorable patientprognosis.
INTRODUCTION
TN3 is an extracelluhar matrix glycoprotein with a unique
six-armed macromolecular structure, which is known to bc an
essential factor for modulation of reciprocal interactions be-
tween the epithehium and mesenchyme during embryogenesis
(1-3). A number of studies have demonstrated prominent TN
Received 2/28/95; revised 4/14/95; accepted 5/1/95.
I This research was supported in part by Grants-In-Aid from the Mm-
istry of Education, Science and Culture, Japan.
2 To whom requests for reprints should be addressed.3 The abbreviations used are: TN, tenascin; ER, estrogen receptor; TBS,
Tris-buffered saline: DIG, digoxygenin.
expression in human cancers including those of brain (4), colon
Fig. I TN immunohistochemistry in human breast cancer using the mAb RCB-1. Positive TN staining is widely distributed in the stroma ofinfiltrating ductal carcinoma (a). In another case, TN staining is not shown in the tissue (b). The cytoplasm of some cancer cells shows theintense positive reaction (c). TN-positive cancer cells are often located in the margin of cancer nests (6). Bar in d, 200 p.m for a, b, and d, and100 p.m for c.
aThe patients were divided into the following three groups according to the results of TN immunohistochemistry: CaC(cancer cell TN)(+)/
St(stroma TN)(+), CaC(-)/St(+), and CaC(-)/St(-).b The P value in the upper row for each parameter is that for the comparison between CaC(+)/St(+) and CaC(-)/St(-), and that in the lower
row is that for the comparison between Ca(-)/St(+) and CaC(-)/St(-).C ER levels were not available for 12 patients.
37#{176}Cfor 15 mm. To remove unhybridized RNA, the sections
were incubated with 20 p.g/ml RNase A in 1 X STE at 37#{176}Cfor
30 mm, followed by one washing in 1 X STE at 37#{176}Cfor 10 mm,
twice in 2X SSC at 50#{176}Cfor 10 mm, and twice in 0.2X SSC for
10 mm at 50#{176}C.In situ hybridization signals were detected
immunohistochemicably with alkaline phosphatase-conjugated
anti-DIG antibody used according to the manufacturer’s instruc-
Fig. 3 In situ hybridization of human breast cancers using a DIG-labeled cRNA probe for TN mRNA. In an infiltrating ductal carcinoma, a large
proportion of the stromal cells show positive signals for TN mRNA, but the cancer cells are not labeled (a). Immunoreactive TN is densely depositedin the stroma (b). Another carcinoma shows the cancer cells with positive signals for in situ hybridization (c), and the cells are also labeled by TNimmunohistochemistry (d, arrows). Bar in d, 100 p.m.
Clinical Cancer Research 1039
ment (P = 0.043), c-erbB-2 positivity (P = 0.001), and ER
negativity (P = 0.007). The cancer cell TN-positive patients
showed a tendency toward larger tumor diameter and later
clinical stage (P = 0.067). Stromal TN was also correlated with
lymph node metastasis (P = 0.038), but showed no significant
correlation with other characteristics. Fig. 2 shows the survival
curves for the 210 patients. The cancer cell TN(+) group
showed shorter survival than the cancer cell TN(-) group
having either stromal TN(+) status (P < 0.05) or stromal
TN(-) status (P < 0.001). The stromal TN(+) group also
showed a poorer outcome than the TN(-) group (P < 0.05).
In Situ Hybridization. Nine fresh cancer tissues were
examined for the expression of TN mRNA by in situ hybrid-
ization. In six cases, various amounts of mesenchymal cells
expressing TN mRNA were seen in the cancer stroma (Fig. 3a).
TN immunohistochemistry showed stromal staining in these
specimens (Fig. 3b). In four of nine cases, TN mRNA was
identified in cancer cells often located at the periphery of cancer
nests adjacent to the mesenchyme (Fig. 3c). In two of these four
cases, the cytoplasm of the cancer cells was positive for immu-
noreactive TN (Fig. 3d, arrows). This result confirmed that
immunohistochemical TN staining of cancer cells denotes TN
production by the cells themselves.
DISCUSSION
In breast cancers, an elevation of c-erbB-2 protein bevels
and this gene amplification have been found to be associated
with increased tumor grade (31-34). The ER status of the breast
cancer cells is correlated well with the response to hormonal
treatment and to some extent with the risk of recurrence, and
thus overall survival (35, 36). Each of these two factors is
accepted as a possible prognostic marker in human breast can-
cer, but it is generally agreed that they do not correlate well with
each other (31). Comparison of the results for these two param-
eters suggest that c-erbB-2 is much more effective than ER and
a rather independent factor (37). In this study, the possible
utility of TN as a prognostic marker of breast cancers was
examined. Seventy-seven of 210 breast cancers showed stromal
staining for TN; the survival of the patients was poorer than that
in the other 133 stromal TN(-) patients. Among these 77 cases,
we found 12 also showing TN(+) cancer cells; the 5-year
survival of these patients was extremely poor. Thus, we identi-
fled TN to be a prognostic marker, especially when it is ex-
pressed in cancer cells, which permits the discrimination of a
subgroup at a high risk of early relapse among breast cancer
patients. However, we found that many of these cases were ER
negative and c-erbB-2 positive. There was also a significant
relationship of cancer cell TN positivity to lymph node metas-
tasis. Therefore, TN expression of cancer cells may not be an
independent parameter.
It is generally accepted that the mesenchymal cells are the
cellular source of TN (1, 14, 38, 39). Also, a few investigators
have reported TN expression in the epithelium. In chick devel-
oping hung, in situ hybridization demonstrates TN mRNA in the
most distal epithelial cells of the bronchi as well as the adjacent
fibroblasts (40, 41). During chick feather morphogenesis, TN
mRNA is detected initially in the overlying epithelia and then in
the underlying mesenchyme (42). In these developing tissues,
however, immunoreactive TN is positive in the basement mem-
brane and/or the surrounding mesenchyme, but not in the
epithelia. The results suggest that TN synthesized by the
epithehium can be released promptly into the extracellular
space and deposited there. Immunohistochemistry of cancer
tissues, including comedo carcinomas of the breast (16),
metastasized prostatic carcinoma in a lymph node (13), and
squamous cell carcinomas and adenocarcinomas of the lung
(9), has demonstrated cytoplasmic TN staining, suggesting
that cancer cells also produce TN. The present results ac-
quired by in situ hybridization clearly demonstrated TN
synthesis by cancer cells as well as by the stromal cells. Four
of the nine cases were positive for TN mRNA, but immuno-
reactive TN was found in cancer cells in only two of them.
TN produced by cancer cells may be also released promptly
into the extracellular space, but a small amount, probably
caused by extreme overexpression or retardation of its re-
lease, is retained in the cancer cell cytoplasm.
Of the various proposed functions of TN, antiadhesion
would seem to be the most probable (43, 44). Fibroblast attach-
ment to fibronectin-coated plates is inhibited by TN addition to
the culture medium in a concentration-dependent manner (45).
TN released from cancer cells may loosen adhesions between
the cells and adhesive extracellubar matrix proteins, such as
fibronectin, and may increase the mobility of cancer cells, with
resulting increased invasive capacity. The TN function, at least
partially, could account for the relationship between the TN
positivity of the cancer cells and the clinicopathological char-
acteristics, such as the tendency to larger tumor size and the
significantly elevated incidence of lymph node metastasis. We
also found that the outcome associated with TN(+) stroma was
poor compared with TN(-) stroma, and that lymph node me-
tastasis occurred more frequently in association with TN(+)
stroma than with TN(-) stroma. According to this result, stro-
mal TN in the cancer tissues may promote cancer spreading. Our
study also indicates that the stromal TN detected by immuno-
histochemistry could have been secreted from both the cancer
cells and the stromal cells, a state which may make it more
complicated to determine TN functions in cancer stroma. There-
fore, it is necessary to further clarify the TN-secreting cells and
the timing of deposition during tumor progression.
ACKNOWLEDGMENTS
We are grateful to A. Kamimori of Matsusaka Chuo General
Hospital for technological assistance.
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1995;1:1035-1041. Clin Cancer Res A Ishihara, T Yoshida, H Tamaki, et al. breast cancer and its prognostic significance.Tenascin expression in cancer cells and stroma of human