HMGA1 Protein Overexpression in Human Breast Carcinomas: Correlation with Erbb2 Expression

Vol. 10, 7637–7644, November 15, 2004 Clinical Cancer Research 7637

HMGA1 Protein Overexpression in Human Breast Carcinomas: Correlation with ErbB2 Expression

Gennaro Chiappetta,1 Gerardo Botti,1 cases of grade 3 breast ductal carcinomas and 7 cases of Mario Monaco,1 Rosa Pasquinelli,1 breast lobular carcinomas. We found that HMGA1 expres- 2 1 sion tended to be associated only with c-erbB-2 expression ؍ ,Francesca Pentimalli, Maurizio Di Bonito 1 2 (Spearman rho: 0.36; P 0.065). Taken together, these Giuseppe D’Aiuto, Monica Fedele, results suggest that HMGA1 expression might be a novel 3 4 Rodolfo Iuliano, Emiliano A. Palmieri, indicator for the diagnosis and prognosis of human breast Giovanna Maria Pierantoni,2 Vincenzo Giancotti,5 cancer. and Alfredo Fusco2 1Istituto Nazionale dei Tumori di Napoli, Fondazione Senatore Pascale, Naples; 2Istituto di Endocrinologia ed Oncologia INTRODUCTION Sperimentale del Consiglio Nazionale delle Ricerche, c/o The HMGA protein family is constituted by proteins Dipartimento di Biologia e Patologia Cellulare e Molecolare, HMGA1a, HMGA1b, and HMGA2. The first two are encoded 3 Universita`di Napoli Federico II, Naples; Dipartimento di Medicina by HMGA1 through alternative splicing (1, 2), whereas HMGA2 Sperimentale e Clinica, University of Catanzaro, Catanzaro; 4Dipartimento di Medicina Clinica, Scienze Cardiovascolari ed is encoded by HMGA2 (3). By interacting with the transcription Immunologiche, Universita`di Napoli Federico II, Naples; and machinery, HMGA1 proteins alter the chromatin structure and 5Dipartimento di Biochimica, Biofisica e Chimica delle thereby regulate the transcriptional activity of several genes (4). Macromolecole, Universita`di Trieste, Trieste, Italy HMGA proteins seem to play their major physiologic role during embryonic development. In fact, HMGA1 expression is ABSTRACT very high during embryogenesis and negligible in normal adult tissues (5, 6). HMGA1 overexpression was first documented in We measured, by immunohistochemistry, HMGA1 pro- transformed thyroid cells (7, 8), then in experimental and human tein expression in 212 breast tissue specimens: 6 normal carcinomas (9), and subsequently in human thyroid (10, 11), samples, 28 hyperplastic lesions (13 with cellular atypia), 11 colorectal (12–14), pancreatic duct cell (15), uterine (16), and fibroadenomas, 10 in situ ductal carcinomas, 144 ductal ovarian (17) cancers. carcinomas, and 13 lobular carcinomas. HMGA1 was not HMGA overexpression is a feature of tumorigenesis and expressed in normal breast tissue; HMGA1 staining was not merely a result of cell transformation. In fact, when rat intense in 40% of hyperplastic lesions with cellular atypia thyroid cells, whose HMGA gene expression is blocked by an and in 60% of ductal carcinomas and weak in fibroadeno- antisense HMGA1 or HMGA2 cDNA construct, are infected by mas and in hyperplastic lesions without cellular atypia. Be- transforming murine retroviruses, they are not able to grow in cause HMGA1 expression was similar among ductal breast soft agar or to form tumors when injected into athymic mice (18, carcinomas with different histologic grading, we evaluated 19). In contrast, untransfected rat cells readily form colonies in the association between HMGA1 expression and that of soft agar after retroviral infection and are highly tumorigenic on other markers of breast carcinoma invasion (estrogen and transplantation. Moreover, HMGA1 is abundantly expressed in progesterone receptors, Ki-67 antigen, and ErbB2) in 21 several human carcinoma cell lines, and blockage of its synthesis by infection with an adenovirus carrying HMGA1 antisense sequences suppressed their growth (20). Breast cancer is the most common cancer and the second Received 2/25/04; revised 6/10/04; accepted 7/2/04. leading cause of cancer mortality in women with about one in Grant support: The “Progetto Speciale Oncosoppressori” Associazione nine being affected during their life. Neoplastic breast diseases Italiana Ricerca sul Cancro, the Programma Italia-USA sulla Terapia dei range from benign fibroadenoma to very aggressive undifferen- Tumori coordinated by Professor Cesare Peschle, the Progetto Finaliz- tiated carcinoma. Growth factors and their receptors, intracellu- zato “Biotecnologie” of Consiglio Nazionale delle Ricerche, the Min- istero dell’Universita`e Ricerca Scientifica e Tecnologica Project “Tera- lar molecules, regulators of cell cycling, and proteases have all pie antineoplastiche innovative,” the “Ministero della Sanita`,” the been shown to be altered in sporadic breast cancer (21) and Universita`degli Studi di Trieste, the Ministero della Ricerca Scientifica ErbB2 overexpression, and a loss of estrogen receptors (ERs), e Tecnologica “Piani di Potenziamento della Rete Scientifica e Tecno- correlates with a poor prognosis. logica,” and the Associazione Partenopea per la Ricerche Oncologiche. The aim of our study was to investigate whether HMGA1 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked protein expression might be an indicator for the diagnosis and advertisement in accordance with 18 U.S.C. Section 1734 solely to prognosis of human breast cancer. This possibility was well indicate this fact. supported by previous studies showing that high levels of Requests for reprints: Alfredo Fusco, Dipartimento di Biologia e HMGA1 mRNA are directly correlated with metastatic progres- Patologia Cellulare e Molecolare, Universita`di Napoli Federico II, via S. Pansini 5, 80131 Naples, Italy. Phone: 39-81-7463056; Fax: 39-081- sion among several mouse mammary tumor cell lines (22). 7463037 or 7701016; E-mail: [email protected]. Moreover, both in vitro and in vivo HMGA1b protein binds to ©2004 American Association for Cancer Research. and inhibits the activity of both human and mouse BRCA1

promoters and that an inverse correlation between HMGA1 and proportion of malignant cells stained was scored from 0 to 3: 0, BRCA1 mRNA and protein expression was found in human no stained cells; 1ϩ, Ͻ10% of weak, focal membrane staining mammary carcinoma cell lines and tissues (23). Furthermore, it of cancer cells; 2ϩ, moderate, completely staining of membrane has been demonstrated that human breast epithelial cells har- of cancer cells; and 3ϩ, strong completely staining of mem- boring tetracycline-regulated HMGA1 transgenes acquire the brane in Ͼ10% of cancer cells (Food and Drug Administration ability to form both primary and metastatic tumors in nude mice approved). only when the transgenes are actively expressed (24). All of Statistical Analysis. The Fisher’s exact test was used to these data would suggest an important role of the HMGA1 analyze intrascore differences of HMGA1 staining among his- overexpression in the process of breast carcinogenesis. tologic types of breast tissue and interhistologic grading differ- In the study reported here, HMGA1 protein was detected in ences of HMGA1-positive cases among ductal breast carcino- 60% of ductal carcinomas and in almost all of the lobular mas. One-way ANOVA was used to analyze interhistologic carcinomas. In addition, HMGA1 expression was similar among grading differences of percent HMGA1-positive cells among ductal breast carcinomas with different histologic grading but ductal breast carcinomas. The nonparametric Spearman corre- tended to be correlated with c-erbB2 amplification. lation was used to analyze the association between HMGA1

MATERIALS AND METHODS Collection of Breast Tissue Samples. A total of 212 breast tissue specimens constituted by 6 normal breast tissues, 28 hyperplastic lesions (13 with atypical cells), 11 fibroadenomas, 10 in situ ductal carcinomas, 144 ductal carcinomas, and 13 lobular carcinomas were collected at the Istituto Nazionale dei Tumori di Napoli (Naples, Italy) from patients undergoing surgery for breast cancer. All tissue samples were fixed imme- diately after surgical removal in 4% paraformaldehyde in PBS. The criteria for inclusion in the study were that the routinely processed paraffin blocks were suitable for immunohistochemistry and adequate clinical information. Immunohistochemical Analysis. Paraffin sections (5 to 6 ␮m) were deparaffinized, placed in a solution of absolute methanol and 0.3% hydrogen peroxide for 30 minutes and then washed in PBS before immunoperoxidase staining. For ER, progesterone receptor (PgR), and Ki-67 staining, slides were incubated overnight at 4°C in a humidified chamber with ER monoclonal antibody (1:50 dilution; Dako A/S, Glostrup, Den- mark), PgR monoclonal antibody (1:50 dilution; Dako), or Ki-67 monoclonal antibody (1:20 dilution; Dako). For ErbB2, the slides were stained with the Dako Herceptin Test kit (Dako), which includes a monoclonal mouse antibody against the cytoplasmic domain of the human ErbB2 protein. For HMGA immunostaining, the antibodies used in this study were raised against the synthetic peptide SSSKQQPLASKQ specific for the HMGA1 proteins (10). They were affinity purified against the synthetic peptide. The slides were subsequently incubated with biotinylated goat antirabbit/antimouse IgG for 20 minutes (Dako LSAB2 System) and then with streptavidin horseradish perox- idase for 20 minutes. For immunostaining, the slides were incubated in 3,3Ј-diaminobenzidine (Dako) solution containing 0.06 mmol/L 3,3Ј-diaminobenzidine and 2 mmol/L hydrogen peroxide in 0.05% PBS (pH 7.6) for 5 minutes. After chromogen development, the slides were washed, dehydrated with alcohol and xylene, and mounted with coverslips with Permount mount- ing medium (Proscitech, Kirwan, Australia). Micrographs were Fig. 1 Specificity of the HMGA1 antibodies. A, nuclear staining in a taken on Kodak Ektachrome film with a photo Zeiss system. For breast ductal carcinoma (original magnification, ϫ200). B, immuno- the clinicopathological study of ER, PgR, Ki-67, and HMGA1 staining of the carcinoma (original magnification, ϫ200) shown in A expression, slides were assigned one of four staining scores: 0, with HMGA1 antibodies preincubated with the peptide against which ϩ Ͻ ϩ antibodies were raised. No immunoreactivity was observed. C,No no stained cells; 1 , 10% of stained cells; 2 ,11to50%of immunoreactivity occurred when the carcinoma shown in panel A was stained positive cells; 3ϩ, 51 to 75% of stained cells; and 4ϩ, immunostained without the primary antibodies (original magnification, 76 to 100% of stained cells. For the ErbB 2 expression study, the ϫ200).

Table 1 HMGA1 protein expression in normal and neoplastic breast tissues by immunohistochemistry. HMGA1 staining score * n (%) Histological type 01ϩ 2ϩ 3ϩ 4ϩ Normal breast tissue (n ϭ 6) 6 (100) 0 (0) 0 (0) 0 (0) 0 (0) Hyperplasias of the ductal epithelium without cellular atypia (n ϭ 13) 9 (70) 2 (15) 2 (15) 0 (0) 0 (0) Hyperplasias of ductal epithelium with cellular atypia (n ϭ 15) 9 (60) 0 (0) 2 (13) 2 (13) 2 (13) Fibroadenomas (n ϭ 11) 6 (55) 3 (27) 1 (9) 1 (9) 0 (0) In situ ductal carcinomas (n ϭ 10) 5 (50) 1 (10) 2 (20) 2 (20) 0 (0) Ductal carcinomas (n ϭ 144) 58 (40) † 16 (11) 28 (20) 30 (21) 12 (8) Lobular carcinomas (n ϭ 13) 1 (8) ‡,§ 1 (8) 3 (23) 5 (38) 3 (23) Ductal ϩ lobular carcinomas (n ϭ 157) 59 (38) ‡,¶ 17 (11) 31 (20) 35 (22) 15 (9) * HMGA1 staining score was as follows: 0, no positive cells; ϩ, positive cells; 1ϩ, Ͻ10% of positive cells; 2ϩ, 11–50% of positive cells; 3ϩ, 51–75% of positive cells; 4ϩ, 76–100% positive cells. N, number of cases. The Fisher’s exact test was used to analyze intrascore differences of HMGA1 staining among breast tissues histological types: †P Ͻ 0.05 and ‡P Ͻ 0.01 versus normal breast tissue; §P Ͻ 0.01 and ¶P Ͻ 0.05 versus hyperplasias of the ductal epithelium without cellular atypia.

staining score and that of ER, ErbB2, Ki-67, and PgR in breast RESULTS ductal and lobular carcinomas. Data were analyzed using stand- We measured the expression of the HMGA1 proteins in ard statistical software (SPSS version 9, Chicago, Illinois). P Ͻ 212 breast tissues (6 normal tissues, 13 hyperplasias with cel- 0.05 was considered significant. lular atypia, 15 hyperplasias without cellular atypia, 11 fibro-

Fig. 2 Immunohistochemical analysis of HMGA1 expression in dysplastic and neoplastic breast tissues. Paraffin sections from dysplastic and neoplastic breast tissues were analyzed by immunohistochemistry with antibodies raised against a specific HMGA1 peptide. A,No immunoreactivity appeared in normal tissue (original magnification, ϫ100) or (B) in epithelial hyperplasia without cellular atypia (original magnification, ϫ200). C, nuclear staining in epithelial hyperplasia without cellular atypia (original magnification, ϫ100). D, intense nuclear and cytoplasmic staining in epithelial hyperplasia with cellular atypia (original magnification, ϫ200). E, weak staining in a fibroadenoma (original magnification, ϫ200). F,no immunoreactivity in a fibroadenoma (original magnification, ϫ100).

Fig. 3 Immunohistochemical analysis of HMGA1 expression in malignant breast neoplasias. A, H&E staining of an in situ breast ductal carcinoma (original magnification, ϫ200). B, immunostaining of in situ ductal carcinoma featured in A showing nuclear staining (original magnification, ϫ100). C, H&E staining of lobular breast carcinoma (original magnification, ϫ100). D, immunostaining of the same lobular breast carcinoma showing nuclear staining (original magnification, ϫ400).

adenomas, 10 in situ ductal carcinomas, 144 ductal carcinomas, 67% of lobular carcinomas, and 51% of ductal ϩ lobular car- and 13 lobular carcinomas) with immunohistochemistry with cinomas.

antibodies raised versus the NH2-terminal region of the Representative results are shown Figs. 2 and 3. Fig. 2, B HMGA1 proteins (10). These antibodies are not able to detect and C, shows epithelial hyperplastic lesions without cellular the product of the other member of the HMGA family that is the atypia, which were negative and positive for HMGA1 expres- HMGA2 gene. sion, respectively. Conversely, Fig. 2D shows a positive epithe- The specificity of the antibody is shown in Fig. 1: a lial hyperplastic lesion with atypical cells; the staining is intense positive nuclear staining was present in a breast carcinoma and is comparable with that observed in most of the ductal sample immunostained with the HMGA1 antibody (Fig. 1A); carcinomas. In Fig. 3, we show two representative carcinomas conversely, no staining was observed when the same carcinoma (one lobular carcinoma and one in situ ductal carcinoma) with sample was stained with antibodies preincubated with the pep- H&E (panels A and C) and immunostaining (panels B and D). tide against which antibodies were raised (Fig. 1B). Equally, Nuclear staining was observed only in some of the neoplastic there was no staining in the absence of the primary antibodies cells of an in situ ductal carcinoma (Fig. 3B), whereas protein (Fig. 1C). HMGA1 was expressed in most of the cells of the lobular breast The data of this analysis are summarized in Table 1. No carcinomas (Fig. 3D). It is noteworthy that in some ductal HMGA1 staining was observed in the normal breast tissues (Fig. carcinomas (as also in Fig. 3B) cytoplasmic and nuclear staining 2A). Conversely, HMGA1 expression was variably detected in was observed. This observation is not new because a similar neoplastic breast tissues (in 30% of hyperplastic lesions without subcellular location was detected in thyroid and colon neopla- cellular atypia, 40% of hyperplastic lesions with cellular atypia, sias (11, 14). Expression of very high levels of HMGA1 protein 45% of fibroadenomas, 50% of in situ ductal carcinomas, 60% of ductal carcinomas, 92% of lobular carcinomas, and 62% of ductal ϩ lobular carcinomas). In particular, HMGA1 expression was significantly more frequent in ductal and lobular carcino- Table 2 HMGA1 protein expression by immunohistochemistry in mas compared with normal breast tissue, either alone (P Ͻ 0.05 grades 1, 2 and 3 ductal breast carcinomas and P Ͻ 0.01, respectively) or in combination (P Ͻ 0.01), and HMGA1-positive % HMGA1-positive in lobular carcinomas compared with hyperplastic lesions with- Histological grade cases * n (%) cells † (mean Ϯ SD) out cellular atypia, either alone or in combination with ductal Grade 1 (n ϭ 45) 29 (64) 18Ϯ15 carcinomas (P Ͻ 0.01 and P Ͻ 0.05, respectively). In addition, Grade 2 (n ϭ 35) 21 (60) 17Ϯ 6 HMGA1 staining was low-to-intermediate (1 to 50% of positive Grade 3 (n ϭ 64) 36 (56) 16Ϯ 3 cells, scores 1ϩ and 2ϩ) in 100% of hyperplastic lesions * The Fisher’s exact test was used to analyze inter-histological without cellular atypia, 80% of fibroadenomas, 60% of in situ grading differences of HMGA1-positive cases among ductal breast P ϭ ductal carcinomas, and 51% of ductal carcinomas, whereas it carcinomas, showing no significant difference ( not significant). † One-way ANOVA was used to analyze interhistological grading was intermediate-to-high (51 to 100% of positive cells, score differences of percentage of HMGA1-positive cells among ductal breast 3ϩ and 4ϩ) in 67% of hyperplastic lesions with cellular atypia, carcinomas, showing no significant difference (P ϭ not significant).

An example is shown in Fig. 4, where three G3 ductal carcinomas (panels A, B, and C) showed an increase in HMGA1 expression from the case in panel A to that in panel C. Taken together, these findings indicate a lack of association between HMGA1 expression and morphologic grading in ductal carcinomas. Therefore, we measured the expression of other indicators of breast carcinoma invasion (ER and PgR receptors, Ki-67, and ErbB2) in 21 ductal and 7 lobular breast carcinomas (Table 3) and analyzed whether their expressions were correlated with that of HMGA1. As shown in Fig. 5, we found that HMGA1 expression tended to be associated only with c-erbB-2 expression (Spearman rho: 0.36; P ϭ 0.065). In Fig. 6 we show a representative case of a breast ductal carcinoma (whose H&E staining is shown in panels A and D) that expressed both the HMGA1 and ErbB2 oncogenic proteins (panels B and C, respectively) and a case of a breast ductal carcinoma negative for the two oncogenic proteins (panels E and F).

DISCUSSION The aim of our study was to verify whether HMGA1 protein might be an indicator for the diagnosis and prognosis

Table 3 HMGA1, ER, PgR, Ki-67, and ErbB2 protein expression in 21 grade 3 breast ductal carcinomas and 7 breast lobular carcinomas Histological Histological type grading ER * PgR * Ki-67 * c-erbB-2 † HMGA1 * Ductal G3 4ϩ 2ϩ 2ϩ 1ϩ 0 Ductal G3 4ϩ 4ϩ 2ϩ 1ϩ 2ϩ Ductal G3 3ϩ 4ϩ 2ϩ 2ϩ 1ϩ Ductal G3 1ϩ 2ϩ 2ϩ 2ϩ 2ϩ Ductal G3 4ϩ 4ϩ 2ϩ 2ϩ 3ϩ Ductal G3 4ϩ 4ϩ 2ϩ 2ϩ 3ϩ Ductal G3 3ϩ 4ϩ 2ϩ 01ϩ Ductal G3 4ϩ 4ϩ 2ϩ 2ϩ 2ϩ Ductal G3 4ϩ 4ϩ 2ϩ 00 Ductal G3 4ϩ 4ϩ 2ϩ 1ϩ 1ϩ Ductal G3 4ϩ 4ϩ 2ϩ 2ϩ 2ϩ Ductal G3 2ϩ 2ϩ 1ϩ 03ϩ Ductal G3 1ϩ 2ϩ 2ϩ 00 Ductal G3 0 0 1ϩ 3ϩ 1ϩ Ductal G3 1ϩ 02ϩ 4ϩ 4ϩ Ductal G3 3ϩ 2ϩ 2ϩ 3ϩ 2ϩ Fig. 4 Immunohistochemical analysis of HMGA1 expression in breast Ductal G3 4ϩ 2ϩ 2ϩ 3ϩ 2ϩ ductal carcinomas (G3). A, immunostaining of a G3 ductal carcinoma Ductal G3 3ϩ 2ϩ 2ϩ 3ϩ 3ϩ with only a few stained cells. B, immunostaining of another G3 ductal Ductal G3 2ϩ 2ϩ 2ϩ 3ϩ 3ϩ carcinoma with more immunostained cells versus the sample shown in Ductal G3 2ϩ 1ϩ 2ϩ 3ϩ 2ϩ A. C, immunostaining of a third G3 ductal carcinoma showing intense Ductal G3 3ϩ 2ϩ 2ϩ 3ϩ 2ϩ immunoreactivity and more immunostained cells versus the samples Lobular G3 4ϩ 4ϩ 2ϩ 1ϩ 3ϩ shown in A and B. Lobular G3 4ϩ 3ϩ 2ϩ 1ϩ 3ϩ Lobular G2 4ϩ 4ϩ 2ϩ 1ϩ 2ϩ Lobular G2 4ϩ 4ϩ 2ϩ 1ϩ 3ϩ Lobular G1 4ϩ 4ϩ 1ϩ 1ϩ 3ϩ in these samples may account for this result. However, it cannot Lobular G3 4ϩ 3ϩ 2ϩ 2ϩ 3ϩ be excluded at all a biological significance of the HMGA1- Lobular G3 2ϩ 4ϩ 1ϩ 00 cytoplasmic localization. * Staining score was, as follows: 0, no positive cells; 1ϩ, Ͻ10% of HMGA1 staining, expressed either as HMGA1-positive positive cells; 2ϩ, 11–50% of positive cells; 3ϩ, 51–75% of positive cases or as percentage of HMGA1-positive cells, was similar cells; 4ϩ, 76–100% positive cells. ϩ Ͻ among ductal breast carcinomas with different histologic grad- † Staining score was as follows: (0, no stained cells; 1 , 10% of weak, focal membrane staining of cancer cells; 2ϩ, moderate, com- ing (Table 2). In fact, several cases of ductal carcinomas with pletely staining of membrane of cancer cells; 3ϩ, strong completely the same histologic grading showed different HMGA1 staining. staining of membrane in Ͼ10% of cancer cells.

Fig. 5 Scatterplots showing relationships between HMGA1 and ER, ErbB2, Ki-67, and PgR staining scores. The nonparametric Spearman correlation was used to analyze the strength of associations between variables.

of human breast cancer. We found that 60% of ductal carci- human breast cancers: rapid proliferation and a poor prognosis nomas and almost all lobular carcinomas expressed HMGA1 of breast cancer are associated with c-erbB2 amplification (21). protein versus none of the normal breast tissues. In addition, It could be hypothesized that HMGA1 proteins would be able to more than half of these HMGA1-positive cases showed an induce c-erbB2 expression acting on its promoter or, alterna- intermediate-to-high staining score. Conversely, HMGA1 ex- tively, that the activation of the ErbB2 transduction pathway pression occurred in a lower percent of benign lesions and may lead to increased HMGA1 protein synthesis. However the hyperproliferative disorders, showing on average a low-to- first hypothesis appears unlikely because of the absence of intermediate staining score. Noteworthy, although HMGA1 AT-rich regions (where the HMGA1 proteins bind DNA) in the protein was detected only in 40% of hyperplastic lesions with c-erbB2 promoter. cellular atypia, its staining score was similar to that of Overexpression of HMGA1 might be critical in the process carcinomas. Similar results were obtained in atypical colon of breast carcinogenesis. In fact, we previously demonstrated adenomas (13, 14), suggesting that the induction of the that HMGA1 negatively regulates the expression of the BRCA1 HMGA1 proteins precedes the appearance of the clearly gene, which is mutated in most familial breast carcinomas, and malignant phenotype. its expression is reduced in the sporadic forms (23). Therefore, There was no association between HMGA1 expression and the induction of HMGA1 expression in breast carcinoma would histologic grading of ductal carcinomas. In fact, HMGA1 stain- down-regulate BRCA1 expression, thereby accounting for the ing differed among malignant neoplastic lesions of the same aggressive phenotype. Moreover, HMGA1 expression might morphologic grading. This prompted us to investigate whether up-regulate several genes that are required for the process of HMGA1 expression was correlated with other prognostic indi- invasion and metastasis (24). cators of breast carcinomas. We found that the staining score for It is interesting that a significant number (30%) of ductal HMGA1 tended to be correlated with that for ErbB2. This is a breast carcinomas did not express the HMGA1 gene. This result transmembrane protein with substantial homology to epidermal appears to contrast with the finding of HMGA1 expression in all growth factor receptor. Gene amplification coupled with result- malignant thyroid, colonic, and pancreatic neoplasias studied ant overexpression of the ErbB2 protein occurs in ϳ25% of thus far (11, 14, 15). Two hypotheses might be envisaged to

Fig. 6 Immunohistochemical analysis of HMGA1 and c-erbB-2 expression in breast ductal carcinomas. A, H&E of a breast carcinoma. (original magnification, ϫ100). B, immunostaining of the breast carcinoma featured in A with HMGA1 antibody showing nuclear and cytoplasmic staining (original magnification, ϫ200). C, immunostaining of the breast carcinoma featured in Fig. 5A with c-erb-B2 antibody showing cytoplasmic staining (original magnification, ϫ200). D, H&E staining of breast carcinoma (original magnification, ϫ100). E, immunostaining of the breast carcinoma featured in D with HMGA1 antibody showing no immunoreactivity (original magnification, ϫ200). F, immunostaining of the breast carcinoma featured in D with ErbB2 antibody showing the absence of immunoreactivity (original magnification, ϫ200).

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Gennaro Chiappetta, Gerardo Botti, Mario Monaco, et al.

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