ANTICANCER RESEARCH 27: 959-964 (2007)

The Prognostic Significance of Human Epidermal Growth Factor Receptor Correlations in Squamous Cell Cervical Carcinoma

I. FUCHS1, N. VORSTEHER2, H. BÜHLER3, K. EVERS2, J. SEHOULI1, G. SCHALLER4 and S. KÜMMEL5

1Department of Gynecology, Charité C. Virchow, Augustenburger Platz 1, 13353 Berlin; 2Department of Gynecology, Charité Campus B. Franklin, Berlin; 3Department of Gynecology, Ruhr-University Bochum, Marienhospital, Hölkeskampring 40, 44625 Herne; 4Breast care institute, Offenbacher Strasse 8, 14197 München; 5Department of Gynecology and Obstetrics, University of Duisburg-Essen, Germany

Abstract. The aim of this study was to investigate the expression The four members of the human epidermal growth factor and prognostic influence of HER1 (EGFR), HER2 (c-erb-B2), receptor (HER) family: HER1 (EGFR, erbB1), HER2 HER3 (c-erb-B3) and HER4 (cerb-B4) in squamous cell cervical (erbB/neu), HER3 (erbB3) and HER4 (erbB4) are carcinomas (SCC) and the importance of receptor correlations. transmembrane tyrosine kinases which take part in the Patients and Methods: 78 SCC were stained immunohisto- regulation of cell proliferation, adhesion, migration and chemically for HER1-HER4. HER2 gene amplification was differentiation (2). Receptor activation and signal determined using fluorescence in situ hybridization (FISH). transduction act through homo- and heterodimerisation. The Parametric correlations were performed between the four receptors formation of various dimeric pairs is dependent on the and tumor characteristics. Overall survival was evaluated by uni- affinity between the different receptors and the concentration and multivariate analyses. Results: Overexpression was found in of both ligands and receptors (3, 4). The specific pattern of 63% of SCC for HER1, in 21.8% for HER2, in 74.4% for HER3 the heterodimeric pairs seems to modulate the intracellular and in 79.5% for HER4. Correlations were observed between response. Receptor overexpression, especially of HER1 and HER1 and HER4 (p=0.019). Survival analyses revealed a HER2 has been associated with malignant potential and poor significant association of HER1 overexpression with favorable prognosis in various tumors (5). outcome (p=0.016), while overexpression of HER2 and HER3 These findings led to the development of specific was associated with poor prognosis (p=0.006; p=0.05, antibodies targeting HER1 and HER2 for modern respectively). HER1 remained significant in multivariate analysis. anticancer therapies (6). The development of pan HER Conclusion: These data suggest that the prognostic relevance of the inhibitors are hoped to further enhance the anti-tumor different HER receptors is influenced by the balance between the effects (7). In breast cancer as well as in other tumors, the various receptors, especially of HER4. anti-HER2 antibody trastuzumab (HerceptinTM,R) significantly improves survival, the strong anti-tumor activity Carcinoma of the uterine cervix remains the leading cause being restricted to HER2-overexpressing tumours (8). of cancer death in young women. In aggressive disease, a In carcinoma of the uterine cervix there are conflicting multimodal treatment approach includes platinum-based results regarding the frequency of HER2 expression, its chemotherapy next to surgery and radiation, however, the prognostic consequence and consequently its value as a efficacy of systemic treatment in this tumor entity is very potential therapeutic target. Even more controversies exist limited (1). The identification of new therapeutic targets is regarding the role of the other EGF receptors in cervical therefore a major goal in improving patient outcome. cancer. This study, therefore, aims to investigate the expression pattern of all four EGF receptors, HER1-HER4, and the influence on prognosis focusing on squamous cell carcinomas of the uterine cervix. Correspondence to: Ilka Fuchs, MD, Department of Gynecology, Charité C. Virchow, Augustenburger Platz 1, 13353 Berlin, Patients and Methods Germany. Tel: +49 30 450664365, Fax: +49 30 450564932, [email protected] Patients. This study included paraffin-embedded tumour samples of 78 patients diagnosed between 1987 and 1994 with primary Key Words: Squamous cell carcinoma, cervical cancer, HER family, invasive squamous cell cervical carcinoma. Age at diagnosis ranged fluorescence in situ hybridization, gene amplification. from 24 to 89 years, with a median of 50 years. Clinical stage was Ia

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Table I. Correlation between HER1-HER4 and tumour characteristics (n=78).

HER1 HER2 HER3 HER4 FIGO

HER2 Correlation 0.108 Significance 0.347 HER3 Correlation 0.087 0.026 Significance 0.448 0.824 HER4 Correlation 0.266 0.037 0.065 Significance 0.019 0.745 0.570 FIGO Correlation 0.063 0.163 0.229 0.153 Significance 0.582 0.154 0.050 0.180 Grading Correlation 0.040 0.066 0.087 0.037 0.193 Significance 0.727 0.565 0.446 0.744 0.090

for one patient, Ib for 33 patients, IIa for six patients, IIb for 16 Figure 1. Overall survival according to HER1 overexpression. Patients with patients, IIIa for four patients, IIIb for 12 patients, IVa for three HER1-positive tumours (interrupted line) had a significantly longer overall survival than patients with HER1-negative tumours (continuous line). patients and IVb for three patients according to the International Federation of Obstetrics and Gynaecology (FIGO) staging system. Treatment consisted of surgery (radical hysterectomy +/- systematic lymph node dissection) in 39 patients, radiotherapy in survival curves according to Kaplan-Meier combined with log-rank- 26 patients and surgery followed by radiotherapy in another 11 tests. For multivariate analyses, Cox regression models were patients. In four patients, treatment was followed by platinum- performed. The level of significance was determined as p<0.05. based chemotherapy. Two patients did not receive any adjuvant treatment: a 84-year-old patient refused curative therapy on the Results basis of age, treatment was omitted for stage IV b disease in another patient due to impaired clinical status. Forty-one patients Of the 78 tumors, a positive HER2 status was found in 17 were classified as grade II and 37 as grade III. Median follow up for overall survival was 60 months (1-180 months). cases (21.8%). Receptor overexpression was found in 49 cases (63%) for HER1, 58 cases (74.4%) for HER3 and in Immunohistochemistry (IHC) and Fluorescence in situ hybridisation 62 cases (79.5%) for HER4. Correlation analysis was (FISH). For a detailed description of the methodology applied in performed between the EGF receptors and between this study please refer to Fuchs et al. (9). classical tumor characteristics (FIGO stage and grading). Each slide was evaluated by two independent examiners who We observed direct correlations between overexpression of were blinded to the staining results of the partner proteins, the HER1 and HER4 (p=0.019). We did not find any clinical data and the evaluation result of the second examiner. For HER2, the official FDA scoring guidelines for predictive significant correlations between the expression profile of assessment in breast carcinoma were applied for the cervical any other EGF pairs or between HER1-4 and FIGO stage carcinomas (HercepTest kit guidelines): 0 for no staining or or grading (Table I). membrane staining in less than 10% of the tumor cells; 1+ for only HER1 overexpression was significantly associated with a partial, weak staining of the cell membrane of more than 10% of better overall survival (Figure 1, p=0.016). The 5-year the tumor cells; 2+ for moderate staining of the complete cell survival rate was 83.7% in HER1-positive patients membrane in more than 10% of the tumor cells; 3+ for intense compared to 58.6% in HER1-negative patients. These staining of the complete membrane in more than 10% of the tumor cells. HER2 overexpression was assessed as negative for scores of differences remained evident after controlling for stage 0 or 1+ and positive 3+. For a score of 2+ the specimens were (p=0.01) and grading (p=0.01). considered undetermined and these cases were, therefore, re- HER2 overexpression in the carcinoma was significantly evaluated by FISH analysis. associated with poor overall survival (Figure 2, p=0.006). HER1, HER3 and HER4 expression was analysed according to The 5-year survival rate was 47.1% in HER2-positive the immunoreactive score (IRS) described by Remmele and patients compared to 82% in HER2-negative patients. These Stegner (10) and was assessed as overexpression for HER3 at a differences remained significant after controlling for stage score >3 and for EGFR and HER4 at a score ≥6. (p=0.03) and grading (p=0.008) in the univariate analysis. Statistical analyses. Statistical analyses were performed using SPSS HER3 overexpression was associated with poor overall 11.0 for Windows (SPSS Inc.). Univariate analyses were assessed survival just below reaching statistical significance (p=0.05, with ¯2 and Fisher’s exact tests. Survival rates were assessed using Figure 3). The 5-year survival rate was 69% in HER3-

960 Fuchs et al: HER Correlations in Cervical Cancer

Figure 2. Overall survival according to the HER2 status. HER2-positive Figure 3. Overall survival according to HER3 overexpression. HER3- patients (interrupted line) had a significantly shorter overall survival than positive patients (interrupted line) had a significantly shorter overall HER2-negative patients (continuous line). survival than HER3-negative patients (continuous line).

positive compared to 90% in HER3-negative patients. Table II. Multivariate Cox regression model with overall survival as These differences were lost after controlling for FIGO stage primary endpoint. (p=0.42) and grading (p=0.065). Variable Exp (B) 95% CI p-value HER4 overexpression itself did not show any statistically significant association with prognosis (p=0.2). HER1 0.3 0.12-0.86 0.025 Multivariate analysis including FIGO stage and grading HER2 2.3 0.89-5.97 0.087 proved FIGO stage to be the most important prognostic HER3 2.5 0.23-11.68 0.226 factor, but confirmed HER1 to be associated with a good HER4 1.2 0.23-6.09 0.848 FIGO stage 11.5 3.30-39.94 0.001 prognosis (Table II). Grading 1.0 3.67-2.56 0.954 Discussion

In contrast to various other tumours, in cervical carcimoma This rate is comparable with that observed in breast cancer. specific antibody therapy targeting the HER family has not The results reflect the observation of previous studies in entered clinical practice. This is mainly due to the that firstly squamous cell carcinomas express lower HER2 substantial disagreement regarding the rate of HER levels than adenocarcinomas (12, 13) and secondly that the overexpression, the expression pattern of the individual positive-rate is generally lower if a standardized scoring receptors and ultimately their prognostic significance. system is applied as compared to non-standardized analyses In preceding reports the HER2 overexpression rate (11). The latter observation is underlined by the fact that in ranged between 3% (11) and 77% (12). These large our study for HER1, HER3 and HER4 lacking an discrepancies have been attributed to methodological established evaluation system the overexpression rates variations, differences in the threshold for the definition of clearly exceeded that of HER2. A very low HER2 HER2 overexpression as well as to diversities of the tumor expression rate of only 3% was described by Chaves Blanco histologies within the same study. et al. (10) in 35 primary cervical cancers. Since they used a In order to overcome these obstacles, we focused on similar methodology, the low expression rate might be due squamous cell carcinomas as a single histological tumor type to the limited study size and their inclusion of various tumor and adopted the scoring system applied for the predictive types with unknown differentiation and grading. HER2 assessment in breast cancer. HER2 positivity was It is noteworthy that in our study the various HER stated in cases with strong HER2 overexpression or in cases receptors had a different prognostic impact. Similar to the with moderate expression and concomitant HER2 gene findings in breast cancer, HER2 and HER3 overexpression amplification: A positive HER2 status was found in 21.7 %. was associated with a poor prognosis (8, 9, 14). In contrast,

961 ANTICANCER RESEARCH 27: 959-964 (2007) overexpression of HER1 was associated with a favorable References prognosis. Though no prognostic impact was found for HER4 overexpression itself, we found a significant 1 Pecorelli S, Pasinetti B, Angioli R, Favalli G and Odicino F: correlation between HER1 and HER4. Systemic therapy for gynecological neoplasms: ovary, cervix, and In the literature, the prognostic relevance of the EGF endometrium. Cancer Chemother Biol Response Modif 22: receptors is mainly based on the evaluation of HER1 or 515-544, 2005. 2 Yarden Y and Sliwkowski MX: Untangling the ErbB signalling HER2 with - at first sight - contradictory results: several network. 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Anticancer Res 26: 4397-4402, 2006. Firstly, all four EGF receptors can be found in squamous 10 Remmele W and Stegner HE: Recommendation for uniform definition of an immunoreactive score (IRS) for cell carcinomas of the uterine cervix. Secondly, the rate of a immunohistochemical estrogen receptor detection (ER-ICA) in positive HER2 status in this tumor entity seems to be breast cancer tissue. Pathologe 8: 138-140, 1987. similar to that found in breast cancer. Thirdly, the specific 11 Chavez-Blanco A, Perez-Sanchez V, Gonzalez-Fierro A, Vela- function of the different EGF receptors and consequently Chavez T, Candelaria M, Cetina L, Vidal S and Duenas- their prognostic relevance is closely connected to the Gonzalez A: HER2 expression in cervical cancer as a potential expression profile of the other HER members, especially therapeutic target. BMC Cancer 4: 59-65, 2004. that of HER4. Transferring these observations into clinical 12 Kihana T, Tsuda H, Teshima S, Nomoto K, Tsugane S, Sonoda T, Matsuura S and Hirohashi S: Prognostic significance of the practice, squamous cell carcinoma of the uterine cervix overexpression of c-erbB-2 protein in adenocarcinoma of the represents a worthwhile target for directed anti-HER uterine cervix. Cancer 73: 148-153, 1994. antibody therapy. However, it becomes increasingly evident 13 Lee JS, Kim HS, Jung JJ, Lee MC and Park CS: Expression of that not only the single growth factor receptor, but also the vascular endothelial growth factor in adenocarcinomas of the expression profiles of the individual HER members, uterine cervix and its relation to angiogenesis and p53 and c- especially of HER4, should be taken into consideration. erbB-2 protein expression. Gynecol Oncol 85: 469-475, 2002. Further prospective studies on the prognostic effects of 14 Witton CJ, Reeves JR, Going JJ, Cooke TG and Bartlett JM: Expression of the HER1-4 family of receptor tyrosine kinases HER receptor combinations are urgently needed. in breast cancer. J Pathol 200: 290-297, 2003. 15 Califano D, Losito S, Pisano C, Santelli G, Greggi S, Iodice F, Acknowledgements DiVagno G, Silvestro G, Tambaro R, Formato R, Iaffaioli VR, Di Maio M and Pignata S: Significance of erb-B2 immuno- We thank J. Pachaly for his invaluable help in statistical matters. reactivity in cervical cancer.Front Biosci 11: 2071-2076, 2006.

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