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From the Departments of Pathology*
and InternalMedicine,
Division of Hematology and Oncology,and the Comprehensive Cancer Center,
University of Michigan, Ann Arbor, Michigan
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Abstract |
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RhoC-GTPase is a member of the Ras-superfamily of small guanosine triphosphatases (GTPases). Activation of Rho proteins leads to assembly of the actin-myosin contractile filaments into focal adhesion complexes that lead to cell polarity and facilitate motility.4-6 Our laboratory has detected overexpression of RhoC mRNA in advanced breast cancers by in situ hybridization, and subsequently characterized RhoC as a transforming oncogene for human mammary epithelial cells, whose overexpression results in a highly motile and invasive phenotype that recapitulates the most lethal form of locally advanced breast cancer, inflammatory breast cancer.
We hypothesized that, given the known functions of the RhoC proteins, RhoC expression would be a good marker to identify breast cancer patients with highly aggressive and motile tumors and guide therapeutic interventions before the development of metastases. Immunohistochemistry is a reproducible and technically simple procedure that would allow testing for RhoC protein expression in the clinical setting. We set out to characterize the expression of RhoC protein in normal, benign, premalignant, and malignant breast disease, with special focus on small (<1 cm) invasive carcinomas with high metastatic potential and/or known metastases.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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We evaluated 182 specimens from 164 patients. Breast tissues were obtained from surgical resections and biopsies from the breast and sites of distant metastases. These cases were selected from the surgical pathology files at the University of Michigan, reviewed by the study pathologist (CGK), and placed in the following pathological categories: normal breast parenchyma (5 cases), fibrocystic changes (5 cases), fibroadenomas (3 cases), atypical ductal hyperplasia (7 cases), ductal carcinoma in situ (11 cases), invasive ductal carcinoma (114 cases), other types of invasive carcinoma (lobular, 13 cases; mucinous, 6 cases; medullary, 2 cases). In addition, 16 metastatic deposits were analyzed, 9 of which had their corresponding primary tumor to compare. Invasive carcinomas were subdivided by stage into stages I, II, III, and IV. Hormonal receptor status and immunohistochemical staining for HER2/neu was available for most patients. Clinical follow-up information was available for all patients. Patient identifiers were removed for subsequent analyses.
Development of RhoC-Specific Antibody
Because RhoC-GTPase has high homology to other members of the Rho
family, RhoA and RhoB, both at the cDNA and the protein level, most
available antibodies are cross-reactive with RhoA, RhoB, and RhoC.
To attempt to develop an antibody specific for RhoC and not for other
Rho family members, a peptide representing a unique epitope was
synthesized at the University of Michigan Protein Core. The
C-terminal region peptide (GLVQVRKNKRRRGCPIL) was chosen because of its
uniqueness and antigenic potential. After injection in rabbits, immune
sera were obtained following standard techniques. Western blot
confirmed the specificity of the antibody for RhoC (Figure 1)
. Specifically no cross-reaction was
observed to recombinant RhoA. To further prove the high specificity of
the antibody for RhoC protein, we performed a competition assay by
incubating the anti-RhoC antibody with increasing concentrations of the
RhoC peptide in 6 ml of 0.3% bovine serum albumin for 6 hours at
4°C. Subsequently a Western blot was performed following standard
procedures (Figure 2A)
. As illustrated in
Figure 2B
, the specificity of the antibody was also checked by blocking
the binding by incubating overnight with a 10-fold molar excess of the
RhoC peptide.
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Immunohistochemistry was performed on formalin-fixed, paraffin-embedded tissue sections that were cut 4-µm thick and stained with polyclonal anti-RhoC antibody. The antibody was titered and used at a 1:1500 dilution for 30 minutes at room temperature, with no previous antigen retrieval. The detection reaction followed the DAKO Envision+ System peroxidase kit protocol (DAKO, Carpinteria, CA). Diaminobenzidine was used as chromogen and hematoxylin was used as counter stain. As positive controls we used tumor xenografts from a cell line known to overexpress RhoC (SUM 149) and from human mammary epithelial cells transfected with RhoC, and patient tumor specimens previously demonstrated to overexpress RhoC by in situ hybridization.7 Negative controls were done by omitting the primary antibody.
Interpretation of Stains
Because RhoC protein interacts with the contractile cytoskeleton
of the cell and is localized to the submembrane space, cytoplasmic
stain was expected. Not surprisingly, myoepithelial cells and vascular
smooth muscle cells were strongly positive in all cases, serving as
consistent internal positive controls (Figure 3)
. The intensity of cytoplasmic staining
was scored as 0 to 3+, by comparison to the positive internal controls.
This scoring system has been previously validated.7,8
Diffuse, moderate to strong cytoplasmic staining characterized
RhoC-positive cells (scores 2+ and 3+) (Figure 3; C, D, and E
).
RhoC-negative cells were devoid of any cytoplasmic staining or
contained faint, equivocal staining (scores 0 and 1+) (Figure 3, B and F)
.
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The chi-square test was used to assess differences in RhoC expression between invasive carcinoma of different stages. Fisher’s exact test was used to study the relationship between RhoC expression and development of metastases, to study whether RhoC expression was significantly different in inflammatory breast cancer (IBC) primary tumors versus lymphatic emboli, and to determine the association between RhoC expression and estrogen receptor, progesterone receptor, and HER2/neu status.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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We studied five cases of normal breast parenchyma and five cases
of fibrocystic changes obtained from reduction mammoplasties and breast
biopsies, respectively. RhoC was not detectably expressed in the ductal
epithelium in any cases (Figure 3A)
. In addition, the three
fibroadenomas tested revealed no RhoC protein expression. No RhoC
protein expression was seen in any cases of atypical ductal hyperplasia
(seven cases) low-grade ductal carcinoma in situ (6 cases),
or high-grade ductal carcinoma in situ (five cases) (Figure 3B)
. All cases however had consistent and strong RhoC staining of
myoepithelial cells and vascular smooth muscle, which served as
internal positive control (Figure 3)
.
RhoC Is Expressed in Invasive Carcinomas with Metastases and its Expression Increases with Primary Tumor Stage
Moderate and strong RhoC protein expression (scores 2+ and 3+) was
detected in 36 of the 114 (32%) primary invasive ductal carcinomas.
When invasive ductal carcinomas were categorized by stage, a strong
correlation was found between RhoC protein expression and tumor stage
(chi-square test, P = 0.01) (Figure 4)
. The relationship of RhoC expression
to the development of metastases is illustrated in Figure 5
. Of the 36 invasive ductal carcinomas
that expressed RhoC, 30 (83%) had axillary lymph node or distant
metastases, and 6 (17%) did not metastasize. The specificity and
sensitivity of RhoC in predicting the development of metastases was
88% and 47%, respectively. The positive and negative predictive
values were 83% and 56%, respectively.
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. Regardless of the pathological tumor stage, RhoC
expression was not detected in primary invasive lobular carcinoma,
invasive typical medullary carcinomas, or invasive mucinous (colloid)
carcinomas. RhoC Expression in Small Invasive Carcinomas with High Metastatic Potential
When invasive ductal carcinomas were separated by primary tumor
size, 20 tumors (18%) were smaller than 1 cm (Figure 6)
. Of these, 13 had no metastases, 6
metastasized to axillary lymph nodes, and 1 developed metastases to
axillary lymph nodes and colon. RhoC was moderately (2+) expressed in 3
of 7 (43%) tumors that metastasized and not expressed in 12 of 13
(92%) invasive carcinomas that did not metastasize (Fisher’s exact
test P = 0.10) (Figure 6)
. RhoC had a specificity of
92% and a sensitivity of 43% in detecting tumors that have metastatic
ability. RhoC’s positive and negative predictive values were 75%.
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Of the 14 distant metastases [liver
(n = 3), cerebellum (n =
1), bone (n = 4), bone marrow
(n = 1), lung (n = 2),
large intestine (n = 2), ovary
(n = 1), and uterus (n =
1)], 7 (50%) expressed RhoC protein (Figure 2E)
. After these cases
were categorized by histological type, RhoC was expressed in five of
eight (62.5%) metastases from invasive ductal carcinomas, in two of
five (40%) metastases from invasive lobular carcinomas, and it was
negative in the one metastasis from a medullary carcinoma.
RhoC Expression Is Associated with Negative Progesterone Receptor and Overexpression of HER2/neu
Table 1
summarizes the results. We
observed that tumors that expressed RhoC were more frequently negative
for progesterone receptor, and overexpressed Her-2/neu by
immunohistochemistry. Although the associations did not reach
statistical significance they suggest that RhoC expression seems to be
associated with well-known predictors of patient outcome. No
association was found between RhoC and estrogen receptor expression.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Based on these results, we hypothesized that expression of RhoC would identify invasive carcinomas that despite their small size have a highly invasive and metastatic potential, and thus develop into a useful screening tool to be used in the clinical arena. We also hypothesized that RhoC may be a new prognostic marker in patients with breast cancer. To test our hypotheses, we developed a specific and sensitive polyclonal antibody directed against the RhoC protein that can be used for immunohistochemistry, and set out to characterize RhoC protein expression in a wide spectrum of breast pathology, from normal, benign lesions, premalignant and in situ carcinomas, to invasive carcinomas of the breast.
From our results several important conclusions can be drawn. First, RhoC expression may not be an early event in the development of non-IBC breast cancer, but a later genetic alteration that occurs once the cancer cells have acquired invasive capabilities. We showed that RhoC is exclusively expressed in invasive carcinomas and not in normal breast, atypical intraductal hyperplasia, or ductal carcinoma in situ. In IBC, the most lethal type of locally advanced breast cancer that is highly metastatic from its inception, RhoC seems to occur early in its development because 80% of all primary IBCs expressed the protein. These results support our previous observations that RhoC is consistently overexpressed in IBC.9 Interestingly, none of the dermal lymphatic tumor emboli expressed RhoC. A possible explanation may be that endolymphatic tumor emboli are cohesive clumps of cancer cells and do not need to acquire motile capabilities until they reach the site of metastases, at which point the tumor cells extravasate and invade new tissues. This argument is supported by a previous study from our laboratory that showed that intralymphatic tumor emboli strongly express E-cadherin, an epithelial cell-cell adhesion molecule that enables the cancer cells to form tightly cohesive tumor emboli.11
Second, RhoC seems to be a marker of metastatic potential in breast cancer. We demonstrated that nearly half of the invasive ductal carcinomas that developed metastases expressed RhoC (30 of 64 cases, 47%), in contrast to very few of the invasive carcinomas without metastases (6 of 50 cases, 12%). RhoC demonstrated a high specificity (88%) in detecting which tumors have the ability to metastasize. These results are in concordance with previous data showing that overexpression of RhoC-GTPase in immortalized human mammary epithelial cells leads to a motile and invasive phenotype able to develop highly metastatic tumors when injected in nude mice.10 Not surprisingly, we found that RhoC-GTPase expression increases with the stage of the invasive carcinoma.
Third, our results suggest that RhoC protein detection by IBC may be a useful tool capable of identifying small invasive ductal carcinomas with high propensity to metastasize. Although the number of cases in our study is small, RhoC was highly specific (specificity of 92%) in detecting small invasive carcinomas with metastatic potential. Forty-three percent of invasive carcinomas smaller than 1 cm that developed metastases expressed RhoC protein, in contrast to 8% of the small tumors that did not metastasize. We are currently expanding the number of cases to further define these observations. This potential use of RhoC in the clinical setting may have a profound impact in the management of breast cancer patients. Specifically, detection of RhoC expression may identify patients who will benefit from an axillary lymph node dissection given the high risk of metastases. RhoC expression may also suggest the use of chemotherapy in patients with small invasive carcinomas at high risk of metastases.
Our data revealed an association between RhoC expression, Her-2/neu overexpression, and loss of progesterone receptor in invasive ductal carcinomas, both well-established predictors of patient outcome. Although primary invasive lobular carcinomas did not express RhoC, 40% of their distant metastases expressed the protein, suggesting that RhoC expression may be involved in the metastatic process of this type of invasive carcinoma, but may be a later event than in non-IBC invasive ductal carcinomas. RhoC expression does not seem to play a role in the late stages of other uncommon forms of invasive breast cancer, including medullary and colloid (mucinous) carcinomas.
Because RhoC protein is associated with the contractile cytoskeleton of the cell, it is not surprising that it is detected by immunohistochemistry in myoepithelial cells and in the vascular smooth muscle cells. These two cell types served as consistent and strong (3+) internal positive controls for the antibody and were detected in all cases.
This study is the first examining the expression of RhoC-GTPase protein in a wide spectrum of normal breast and of breast disease. It is clear from the results that RhoC is a specific marker of metastatic disease in patients with breast cancer. Importantly, although our data are preliminary, it appears to identify a subset of patients with small primary tumors and high metastatic potential that would benefit from axillary lymph node staging and/or chemotherapy and that would remain otherwise unrecognized.
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Acknowledgements |
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Footnotes |
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Supported in part by DOD grant DAMD17-00-1-0636 (C.G.K.), NIH grant R01CA77612 (S.D.M.), MUNN award from University of Michigan (C.G.K.), DOD grant DAMD 17-00-1-0637 (K.v.G.), and DOD grant DAMD 17-00-1-0345 (S.D.M.).
Accepted for publication November 1, 2001.
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