ORIGINAL STUDY
Evaluation of TOP2A as a Predictive Marker for Endometrial Cancer With Taxane-Containing Adjuvant Chemotherapy Fuminori Ito, MD,* Naoto Furukawa, MD,Þ and Tokiko Nakai, MDþ
Objective: Paclitaxel plus carboplatin and doxorubicin plus cisplatin are usually selected as adjuvant chemotherapy for endometrial cancer. However, biomarkers that can determine the appropriate chemotherapy regimen are not known. In the present study, we performed a retrospective investigation of the association between TOP2A, HER2 overexpression, and diseasefree and overall survival in patients with endometrial cancer receiving taxane and platinum. Methods: Eligible patients had a diagnosis of endometrial cancer based on histology and treated with an adjuvant chemotherapy regimen comprising taxane-platinum after surgery, and the HER2 and TOP2A status of the endometrial cancer regions was determined. Overall survival and disease-free survival between HER2 status and TOP2A status were estimated by the Kaplan-Meier method and compared using the log-rank test. Results: We identified 56 patients who fulfilled the previously described criteria. Median follow-up was 49 months (range, 18-110 months). HER2-positive tumors were detected in 11 patients (19.6%), and TOP2A-positive tumors were detected in 7 patients (12.5%). Overall survival was not significantly different between patients with HER2-positive tumors and those with HER2-negative tumors, although disease-free survival for patients with HER2-positive tumors was significantly lower than disease-free survival for patients with HER2-negative tumors (P = 0.049). In contrast, patients with TOP2A-positive tumors had significantly lower overall survival than did patients with TOP2A-negative tumors (P = 0.020), and disease-free survival for patients with TOP2A-positive tumors tended to be shorter than for those with TOP2A-negative tumors. Conclusions: Patients with TOP2A overexpression have a worse prognosis compared with those with TOP2A nonexpression, and TOP2A may be a useful biomarker in patients receiving adjuvant taxane-platinum regimens with moderate- to high-risk endometrial cancer. Key Words: Biomarker, Endometrial cancer, Taxane-platinum regimens, TOP2A Received August 12, 2015, and in revised form October 13, 2015. Accepted for publication October 14, 2015. (Int J Gynecol Cancer 2016;26: 325Y330)
n 2008, 287,000 endometrial cancer cases were reported worldwide, and in 2012, 319,000 endometrial cancer cases were reported, demonstrating an increased incidence of
I
endometrial cancer.1 The incidence of endometrial cancer has also increased in Japan and is close to that of invasive cervical cancer.2 Endometrial cancer is diagnosed in more than 65% of
*Department of Obstetrics and Gynecology, Nara Medical University; †Department of Obstetrics and Gynecology, Nara Prefecture
Western Medical Center; and ‡Department of Pathology, Nara Medical University, Nara, Japan. Address correspondence and reprint requests to Naoto Furukawa, MD, Department of Obstetrics and Gynecology, Nara Prefecture Western Medical Center, 1-14-16 Mimuro, Sango-cho, Ikoma-gun, Nara 636-0802, Japan. E-mail: [email protected]. The authors declare no conflicts of interest.
Copyright * 2016 by IGCS and ESGO ISSN: 1048-891X DOI: 10.1097/IGC.0000000000000607 International Journal of Gynecological Cancer
& Volume 26, Number 2, February 2016
Copyright © 2016 by IGCS and ESGO. Unauthorized reproduction of this article is prohibited.
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patients at an early stage.3 On the other hand, 15% of patients with early-stage endometrial cancer will experience a recurrence.4 Adjuvant chemotherapy or radiotherapy is applied to reduce the recurrence rate. In Japan, chemotherapy is used more often than radiotherapy because of the adverse effects of radiotherapy, and paclitaxel and carboplatin (TC) are preferred over doxorubicin and cisplatin (AP) in the regimen for endometrial cancer.5 One phase 3 study reported that paclitaxel, doxorubicin, and cisplatin (TAP) regimen significantly improves overall survival compared with AP in patients with advanced or recurrent endometrial cancer,6 and another phase 3 study reported that TC was noninferior in overall survival to TAP in patients with advanced or recurrent endometrial cancer.7 TAP, however, has more neurotoxic and myelosuppressive effects than AP and TC, so TAP is rarely used worldwide.5 Therefore, TC and AP are usually selected as adjuvant chemotherapy for endometrial cancer. The toxicity profile of TC differs from that of AP, although TC has a more favorable toxicity profile and is more often selected than AP.5 Biomarkers that can determine the appropriate chemotherapy regimen are not known. TOP2A encodes an enzyme with a key role in DNA replication. The gene that encodes TOP2A is located at chromosome 17q 12-q21, in proximity to HER2. TOP2A amplification might be linked to the sensitivity to anthracyclines observed in HER2-positive breast cancer.8Y10 Some studies have also reported an association between HER2 overexpression and poor overall survival in patients with endometrial cancer.11,12 To date, however, no studies have demonstrated an association between TOP2A overexpression and overall survival of patients with endometrial cancer. In the present study, we performed a retrospective investigation of the association between TOP2A, HER2 overexpression, and disease-free and overall survival in patients with endometrial cancer receiving taxane and platinum.
METHODS Patient Selection The present study was conducted in accordance with the principles of the Declaration of Helsinki. Eligible patients received a diagnosis of endometrial cancer based on histology and were treated with an adjuvant chemotherapy regimen comprising taxane-platinum after hysterectomy, bilateral salpingo-oophorectomy, and/or lymphadenectomy and tumor resection, between January 2005 and December 2010 at Nara Medical University. Lymphadenectomy was omitted in patients with grades 1 and 2 endometrioid adenocarcinoma and no myometrial invasion evaluated by magnetic resonance imaging. Eligibility criteria for chemotherapy were myometrium invasion greater than 1/2; a special type of histology, such as endometrioid adenocarcinoma grade 3, clear cell carcinoma, uterine serous papillary carcinoma, or carcinosarcoma; lymph node metastasis; or distant metastasis. Patients with residual tumor after surgery were excluded. Clinical data were collected from the medical records. All endometrial cancer specimens were obtained by surgery, and the HER2 and TOP2A status of the endometrial cancer regions was determined. Tissue microarrays were constructed as follows. We extracted a small cylindrical core from a targeted point of each paraffin-embedded
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tissue block and re-embedded several cores into a single paraffin block, ultimately aligning them to fit on a single glass slide. In the present study, we collected from 2 points per sample, using 3-mm cores. The protocol was approved by the institutional review board of Nara Medical University as no. 960.
Immunohistochemistry Immunohistochemical staining for tissue microarray sections was performed using the polyclonal anti-2/neu (cerbB-2) antibody (1:250 dilution; Dako, Carpinteria, CA) and monoclonal topoisomerase II> antibody (1:400; Clone D10G9; Cell Signaling Technology, Beverly, MA). Immunohistochemistry was performed using formalin-fixed, paraffin-embedded tissue sections. Briefly, 2-Km-thick sections were cut using a microtome, transferred onto adhesive slides, and dried at 60-C for 30 minutes. After incubation with primary antibodies, immunohistochemistry was performed using the Leica BONDMAX systems (Mitsubishi Chemical Medicine Co, Tokyo, Japan) following the manufacturer’s instructions.
Interpretation of Immunohistochemical Staining HER2 staining was analyzed according to the guidelines of the American Society of Clinical Oncology/College of American Pathologists, as follows: a value of 0 represents no immunostaining or weak incomplete membranous staining of less than 10% of tumor cells; 1+, weak incomplete membranous staining of more than 10%; 2+, complete membrane staining that is either nonuniform or weak in intensity, but with obvious circumferential distribution of at least 10% of cells; and 3+, uniform intense membrane staining of more than 30% of invasive tumor cells.13 Scores of 2+ and 3+ were defined as HER2 positive. TOP2A staining intensity was scored as 0 to 3+ based on the percentage of positive cells: 0 (no staining), 1+ (G10%), 2+ (10%-50%), or 3+ (950%). A score of 3+ was defined as TOP2A positive. Two independent pathologists blinded to the clinical data and each other’s opinion scored the expression based on the staining intensity. Differences in opinion by the 2 pathologists were resolved by consensus.
Statistical Analysis All statistical analyses were conducted using SPSS version 17 (SPSS Inc, Chicago, IL). Probability values of less than 0.05 were considered to indicate statistical significance. Overall survival and disease-free survival between HER2 status and TOP2A status were estimated by the Kaplan-Meier method and compared using the log-rank test. The Cox proportional hazard model was used to evaluate univariate and independent multivariate associations of the clinical parameters and immunohistochemical status with overall survival and disease-free survival.
RESULTS We identified 56 patients who fulfilled the previously described criteria. Median follow-up was 49 months (range, 18Y110 months). Patient characteristics are presented in Table 1. Of the 56 patients, 36 patients (64.3%) had endometrioid adenocarcinoma (grade 1, n = 23; grade 2, n = 6; grade 3, n = 7), and 20 (35.7%) had other histologic types. Stage distribution * 2016 IGCS and ESGO
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International Journal of Gynecological Cancer
& Volume 26, Number 2, February 2016 TOP2A as Marker for Endometrial Cancer
TABLE 1. Patients’ characteristics No. of patients Median age, y Median follow-up period, mo Histology
Lymphadenectomy Stage
56 61 (41Y77) 49.4 (17.5Y109.5) Endometrioid adenocarcinoma Mixed carcinoma Clear cell carcinoma Poor differentiated carcinoma Carcinosarcoma Yes No I II III IV
was as follows: 35 (62.5%) stage I, 7 (12.5%) stage II, 12 (21.4%) stage III, and 2 (3.6%) stage IV. A total of 51 patients (91.1%) underwent 6 cycles of paclitaxel (175 mg/m2) followed by carboplatin (area under the curve of 5); 5 patients (8.9%) had other taxane-platinum regimens. HER2-positive tumors were detected in 11 patients (19.6%), and TOP2Apositive tumors were detected in 7 patients (12.5%). Overall survival was not significantly different between patients with HER2-positive tumors and those with HER2-negative tumors, although disease-free survival for patients with HER2-positive tumors was significantly lower than disease-free survival for patients with HER2-negative tumors (P = 0.049; Fig. 1). In contrast, patients with TOP2A-positive tumors had
G1/G2/G3
23/6/7 9 3 2 6 49 7 35 7 12 2
significantly lower overall survival than patients with TOP2A-negative tumors (P = 0.020), and disease-free survival for patients with TOP2A-positive tumors tended to be shorter than for those with TOP2A-negative tumors (Fig. 2). Univariate analysis using the Cox proportional hazard model revealed that HER2 status, lymph node metastasis, lymph vascular space involvement, and stage were significantly associated with disease-free survival (Table 2). Multivariate analysis revealed that independent prognostic factors for disease-free survival were HER2 status and lymph vascular space involvement (P = 0.021 and 0.035, respectively; Table 2). On the other hand, univariate analysis revealed that TOP2A, lymph node metastasis, and stage
FIGURE 1. A, Kaplan-Meier estimates of overall survival by HER2 status. B, Kaplan-Meier estimates of disease-free survival by HER2 status. * 2016 IGCS and ESGO
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& Volume 26, Number 2, February 2016
FIGURE 2. A, Kaplan-Meier estimates of overall survival by TOP2A status. B, Kaplan-Meier estimates of disease-free survival by TOP2A status. were significantly associated with overall survival, whereas multivariate analysis revealed no prognostic factors for overall survival (Table 3).
DISCUSSION In the present study, TOP2A status was not detected as a prognostic factor for disease-free survival, although diseasefree survival for patients with TOP2A-positive tumors tended to be shorter than that for patients with TOP2A-negative tumors. Moreover, overall survival for patients with TOP2Apositive tumors was significantly shorter than that for patients with TOP2A-negative tumors. Based on these findings, TOP2A-positive status may predict a poor prognosis in patients with endometrial cancer receiving taxane-platinum
regimens. The metastasis-free interval in breast cancer patients with high TOP2A gene expression is significantly lower than that in patients with low expression.14 Increased TOP2A immunoexpression significantly correlated with advanced stages and tumor aggressiveness, confirming the potential of TOP2A as a prognostic biomarker for nasopharyngeal carcinoma.15 Moreover, higher expression of TOP2A protein in prostate cancer patients is a strong indicator of a poor prognosis.16 The present study revealed that TOP2A-positive status may be a predictor for poor prognosis in patients with endometrial cancer receiving taxane-platinum regimens. In other words, taxane-platinum regimens may not have a sufficient effect for TOP2A-positive endometrial cancer. Anthracycline-based neoadjuvant chemotherapy regimens
TABLE 2. Analysis of risk factors associated with disease-free survival Multivariate Variables HER2 + vs j TOP2A + vs j Lymph node metastasis + vs j Lymph vascular space involvement + vs j Myometrial invasion 91/2 vs e1/2 Histology type 2 vs type 1 Peritoneal cytology + vs j Lymphadenectomy j vs + Stages III, IV vs I, II
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Univariate
P
95% Confidence Interval
Hazard Ratio
0.049 0.093 0.017 0.023 0.227 0.705 0.197 0.303 0.006
0.021 0.335 0.897 0.035
1.21Y10.24 0.52Y6.73 0.13Y6.17 1.08Y8.07
3.52 1.88 1.14 2.95
0.215
0.53Y16.19
2.57 * 2016 IGCS and ESGO
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& Volume 26, Number 2, February 2016 TOP2A as Marker for Endometrial Cancer
TABLE 3. Analysis of risk factors associated with overall survival Multivariate Variables
Univariate
HER2 + vs j TOP2A + vs j Lymph node metastasis + vs j Lymph vascular space involvement + vs j Myometrial invasion 91/2 vs e1/2 Histology type 2 vs type 1 Peritoneal cytology + vs j Lymphadenectomy j vs + Stages III, IV vs I, II
0.544 0.020 0.004 0.826 0.310 0.342 0.490 0.951 0.009
for breast cancer can significantly improve the pathologically complete response rate for patients with HER2 overexpression and concurrent TOP2A amplification.17 In women with HER2 geneYamplified metastatic breast cancer, overall survival in the TOP2A amplified group is significantly longer than that in the nonYTOP2A-amplified group with anthracycline-based chemotherapy.18 Thus, TOP2A status can be a predictor of anthracycline sensitivity in breast cancer. A large study should be conducted to evaluate the association between TOP2A and tumor suppression for anthracycline in endometrial cancer. In approximately 35% to 40% of patients with breast cancer with HER2 gene amplification, TOP2A is coamplified.19 This incidence of coamplification in endometrial cancer, however, is not established because there are no reports of an association between TOP2A and the effect of doxorubicin for endometrial cancer. In the present study, the frequency of both HER2 and TOP2A overexpression was 3.6% (2/56). In HER2-positive breast cancers in which TOP2A was coamplified, the rates of disease-free survival for the trastuzumab-containing regimens were indistinguishable from the rate with regimens without trastuzumab, but among HER2-positive breast cancers without TOP2A coamplification, a disease-free survival benefit with trastuzumabcontaining regimens is observed.20 On the other hand, no interaction has been detected between TOP2A expression and response to trastuzumab in patients with HER2-positive breast cancer.21 In the present study, HER2 status was detected as a significant prognostic factor for disease-free survival. Many studies report an association between HER2 and uterine serous carcinomas, with the rates of HER2 overexpression ranging from 14% to 80% and the rates of HER2 amplification ranging from 21% to 47%.22 Few studies, however, report an association between HER2 and endometrioid adenocarcinomas, and HER2 overexpression and amplification in endometrioid adenocarcinomas are reported in the range of 1% to 47% and 0% to 38%, respectively.22 Uterine serous carcinomas have a higher rate of HER2 expression compared with endometrioid carcinomas, and grade 3 endometrioid adenocarcinomas have a higher rate compared with grade 1-2 endometrioid adenocarcinomas.11 Several studies report an association between
P
95% Confidence Interval
Hazard Ratio
0.609 0.502
0.19Y16.72 0.12Y76.12
1.79 3.02
0.631
0.15Y23.04
1.85
HER2 overexpression and poor overall survival in patients with uterine serous carcinomas.12,23,24 The association between HER2 status and survival in endometrioid adenocarcinomas, however, remains uncertain. HER2 expression in the presence of amplification correlated with overall survival in a multivariate analysis.11 On the other hand, there was no significant effect of HER2 expression or amplification on survival,25 and HER2 overexpression or HER2 amplification was not associated with progression-free survival or overall survival.26 Novel HER2-based therapy in endometrial cancer has demonstrated good potential, but no significant activity was observed in the GOG-181B phase 2 trial of the single-agent trastuzumab in advanced or recurrent endometrial cancer patients.26 A multiinstitutional randomized phase 2 study (NCT01367002) is currently underway to evaluate carboplatin/paclitaxel with or without trastuzumab in patients with advanced or recurrent HER2-positive uterine serous carcinomas. Limitations of the present study include the small sample size and analysis of only immunohistochemistry. To evaluate the association between TOP2A and survival in endometrial cancer, large studies should be conducted that include analysis of TOP2A amplification in addition to immunohistochemistry. In conclusion, patients with TOP2A overexpression have a worse prognosis compared with those with TOP2A nonexpression, and TOP2A may be a useful biomarker in patients receiving adjuvant taxane-platinum regimens with moderate- to high-risk endometrial cancer. Further studies are needed to evaluate the association between TOP2A status and survival and the effects of anthracycline-containing regimens as adjuvant chemotherapy.
REFERENCES 1. Corpus uteriVestimated incidence, all ages. GLOBOCAN 2012. 2012. Available at: http://globocan.iarc.fr/old/summary_table_ site-html.asp?selection=6172&title=Corpus+uteri&sex= 2&type=0&window=1&africa=1&america=2&asia=3&europe= 4&oceania=5&build=6&sort=0&submit=%C2%A0Execute. Accessed Nov. 8, 2015. 2. Matsuda T, Marugame T, Kamo K, et al. Cancer incidence and incidence rates in Japan in 2006: based on data from 15
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population-based cancer registries in the Monitoring of Cancer Incidence in Japan (MCIJ) project. Jpn J Clin Oncol. 2012;42:139Y147. Aoki D. Annual report of Gynecologic Oncology Committee, Japan Society of Obstetrics and Gynecology, 2013. J Obstet Gynaecol Res. 2014;40:338Y348. Creutzberg CL, van Putten WL, Koper PC, et al. Surgery and postoperative radiotherapy versus surgery alone for patients with stage-1 endometrial carcinoma: multicentre randomised trial. PORTEC Study Group. Post Operative Radiation Therapy in Endometrial Carcinoma. Lancet. 2000;355:1404Y1411. Fotopoulou C, Kraetschell R, Dowdy S, et al. Surgical and systemic management of endometrial cancer: an international survey. Arch Gynecol Obstet. 2015;291:897Y905. Fleming GF, Brunetto VL, Cella D, et al. Phase III trial of doxorubicin plus cisplatin with or without paclitaxel plus filgrastim in advanced endometrial carcinoma: a Gynecologic Oncology Group Study. J Clin Oncol. 2004;22:2159Y2166. Miller D, Filiaci V, Fleming G, et al. Late-breaking abstract 1: randomized phase III noninferiority trial of first line chemotherapy for metastatic or recurrent endometrial carcinoma: a Gynecologic Oncology Group study. Gynecol Oncol. 2012;125:771. Konecny GE, Pauletti G, Untch M, et al. Association between HER2, TOP2A, and response to anthracycline-based preoperative chemotherapy in high-risk primary breast cancer. Breast Cancer Res Treat. 2010;120:481Y489. Scandinavian Breast Group T, Tanner M, Isola J, et al. Topoisomerase IIalpha gene amplification predicts favorable treatment response to tailored and dose-escalated anthracycline-based adjuvant chemotherapy in HER-2/ neu-amplified breast cancer: Scandinavian Breast Group Trial 9401. J Clin Oncol. 2006;24:2428Y2436. O’Malley FP, Chia S, Tu D, et al. Topoisomerase II alpha and responsiveness of breast cancer to adjuvant chemotherapy. J Natl Cancer Inst. 2009;101:644Y650. Morrison C, Zanagnolo V, Ramirez N, et al. HER-2 is an independent prognostic factor in endometrial cancer: association with outcome in a large cohort of surgically staged patients. J Clin Oncol. 2006;24:2376Y2385. Togami S, Sasajima Y, Oi T, et al. Clinicopathological and prognostic impact of human epidermal growth factor receptor type 2 (HER2) and hormone receptor expression in uterine papillary serous carcinoma. Cancer Sci. 2012;103:926Y932. Wolff AC, Hammond ME, Schwartz JN, et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. J Clin Oncol. 2007; 25:118Y145.
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14. Brase JC, Schmidt M, Fischbach T, et al. ERBB2 and TOP2A in breast cancer: a comprehensive analysis of gene amplification, RNA levels, and protein expression and their influence on prognosis and prediction. Clin Cancer Res. 2010;16: 2391Y2401. 15. Lan J, Huang HY, Lee SW, et al. TOP2A overexpression as a poor prognostic factor in patients with nasopharyngeal carcinoma. Tumour Biol. 2014;35:179Y187. 16. de Resende MF, Vieira S, Chinen LT, et al. Prognostication of prostate cancer based on TOP2A protein and gene assessment: TOP2A in prostate cancer. J Transl Med. 2013;11:36. 17. Wang J, Xu B, Yuan P, et al. TOP2A amplification in breast cancer is a predictive marker of anthracycline-based neoadjuvant chemotherapy efficacy. Breast Cancer Res Treat. 2012;135:531Y537. 18. Press MF, Sauter G, Buyse M, et al. Alteration of topoisomerase II-alpha gene in human breast cancer: association with responsiveness to anthracycline-based chemotherapy. J Clin Oncol. 2011;29:859Y867. 19. Tubbs R, Barlow WE, Budd GT, et al. Outcome of patients with early-stage breast cancer treated with doxorubicin-based adjuvant chemotherapy as a function of HER2 and TOP2A status. J Clin Oncol. 2009;27:3881Y3886. 20. Slamon D, Eiermann W, Robert N, et al. Adjuvant trastuzumab in HER2-positive breast cancer. N Engl J Med. 2011;365: 1273Y1283. 21. Gianni L, Norton L, Wolmark N, et al. Role of anthracyclines in the treatment of early breast cancer. J Clin Oncol. 2009; 27:4798Y4808. 22. Buza N, Roque DM, Santin AD. HER2/neu in endometrial cancer: a promising therapeutic target with diagnostic challenges. Arch Pathol Lab Med. 2014;138:343Y350. 23. Slomovitz BM, Broaddus RR, Burke TW, et al. Her-2/neu overexpression and amplification in uterine papillary serous carcinoma. J Clin Oncol. 2004;22:3126Y3132. 24. Santin AD, Bellone S, van Stedum S, et al. Amplification of c-erbB2 oncogene: a major prognostic indicator in uterine serous papillary carcinoma. Cancer. 2005;104:1391Y1397. 25. Grushko TA, Filiaci VL, Mundt AJ, et al. An exploratory analysis of HER-2 amplification and overexpression in advanced endometrial carcinoma: a Gynecologic Oncology Group study. Gynecol Oncol. 2008;108:3Y9. 26. Fleming GF, Sill MW, Darcy KM, et al. Phase II trial of trastuzumab in women with advanced or recurrent, HER2-positive endometrial carcinoma: a Gynecologic Oncology Group study. Gynecol Oncol. 2010;116:15Y20.
* 2016 IGCS and ESGO
Copyright © 2016 by IGCS and ESGO. Unauthorized reproduction of this article is prohibited.
Evaluation of TOP2A as a Predictive Marker for Endometrial Cancer With Taxane-Containing Adjuvant Chemotherapy Fuminori Ito, MD,* Naoto Furukawa, MD,Þ and Tokiko Nakai, MDþ
Objective: Paclitaxel plus carboplatin and doxorubicin plus cisplatin are usually selected as adjuvant chemotherapy for endometrial cancer. However, biomarkers that can determine the appropriate chemotherapy regimen are not known. In the present study, we performed a retrospective investigation of the association between TOP2A, HER2 overexpression, and diseasefree and overall survival in patients with endometrial cancer receiving taxane and platinum. Methods: Eligible patients had a diagnosis of endometrial cancer based on histology and treated with an adjuvant chemotherapy regimen comprising taxane-platinum after surgery, and the HER2 and TOP2A status of the endometrial cancer regions was determined. Overall survival and disease-free survival between HER2 status and TOP2A status were estimated by the Kaplan-Meier method and compared using the log-rank test. Results: We identified 56 patients who fulfilled the previously described criteria. Median follow-up was 49 months (range, 18-110 months). HER2-positive tumors were detected in 11 patients (19.6%), and TOP2A-positive tumors were detected in 7 patients (12.5%). Overall survival was not significantly different between patients with HER2-positive tumors and those with HER2-negative tumors, although disease-free survival for patients with HER2-positive tumors was significantly lower than disease-free survival for patients with HER2-negative tumors (P = 0.049). In contrast, patients with TOP2A-positive tumors had significantly lower overall survival than did patients with TOP2A-negative tumors (P = 0.020), and disease-free survival for patients with TOP2A-positive tumors tended to be shorter than for those with TOP2A-negative tumors. Conclusions: Patients with TOP2A overexpression have a worse prognosis compared with those with TOP2A nonexpression, and TOP2A may be a useful biomarker in patients receiving adjuvant taxane-platinum regimens with moderate- to high-risk endometrial cancer. Key Words: Biomarker, Endometrial cancer, Taxane-platinum regimens, TOP2A Received August 12, 2015, and in revised form October 13, 2015. Accepted for publication October 14, 2015. (Int J Gynecol Cancer 2016;26: 325Y330)
n 2008, 287,000 endometrial cancer cases were reported worldwide, and in 2012, 319,000 endometrial cancer cases were reported, demonstrating an increased incidence of
I
endometrial cancer.1 The incidence of endometrial cancer has also increased in Japan and is close to that of invasive cervical cancer.2 Endometrial cancer is diagnosed in more than 65% of
*Department of Obstetrics and Gynecology, Nara Medical University; †Department of Obstetrics and Gynecology, Nara Prefecture
Western Medical Center; and ‡Department of Pathology, Nara Medical University, Nara, Japan. Address correspondence and reprint requests to Naoto Furukawa, MD, Department of Obstetrics and Gynecology, Nara Prefecture Western Medical Center, 1-14-16 Mimuro, Sango-cho, Ikoma-gun, Nara 636-0802, Japan. E-mail: [email protected]. The authors declare no conflicts of interest.
Copyright * 2016 by IGCS and ESGO ISSN: 1048-891X DOI: 10.1097/IGC.0000000000000607 International Journal of Gynecological Cancer
& Volume 26, Number 2, February 2016
Copyright © 2016 by IGCS and ESGO. Unauthorized reproduction of this article is prohibited.
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Ito et al
patients at an early stage.3 On the other hand, 15% of patients with early-stage endometrial cancer will experience a recurrence.4 Adjuvant chemotherapy or radiotherapy is applied to reduce the recurrence rate. In Japan, chemotherapy is used more often than radiotherapy because of the adverse effects of radiotherapy, and paclitaxel and carboplatin (TC) are preferred over doxorubicin and cisplatin (AP) in the regimen for endometrial cancer.5 One phase 3 study reported that paclitaxel, doxorubicin, and cisplatin (TAP) regimen significantly improves overall survival compared with AP in patients with advanced or recurrent endometrial cancer,6 and another phase 3 study reported that TC was noninferior in overall survival to TAP in patients with advanced or recurrent endometrial cancer.7 TAP, however, has more neurotoxic and myelosuppressive effects than AP and TC, so TAP is rarely used worldwide.5 Therefore, TC and AP are usually selected as adjuvant chemotherapy for endometrial cancer. The toxicity profile of TC differs from that of AP, although TC has a more favorable toxicity profile and is more often selected than AP.5 Biomarkers that can determine the appropriate chemotherapy regimen are not known. TOP2A encodes an enzyme with a key role in DNA replication. The gene that encodes TOP2A is located at chromosome 17q 12-q21, in proximity to HER2. TOP2A amplification might be linked to the sensitivity to anthracyclines observed in HER2-positive breast cancer.8Y10 Some studies have also reported an association between HER2 overexpression and poor overall survival in patients with endometrial cancer.11,12 To date, however, no studies have demonstrated an association between TOP2A overexpression and overall survival of patients with endometrial cancer. In the present study, we performed a retrospective investigation of the association between TOP2A, HER2 overexpression, and disease-free and overall survival in patients with endometrial cancer receiving taxane and platinum.
METHODS Patient Selection The present study was conducted in accordance with the principles of the Declaration of Helsinki. Eligible patients received a diagnosis of endometrial cancer based on histology and were treated with an adjuvant chemotherapy regimen comprising taxane-platinum after hysterectomy, bilateral salpingo-oophorectomy, and/or lymphadenectomy and tumor resection, between January 2005 and December 2010 at Nara Medical University. Lymphadenectomy was omitted in patients with grades 1 and 2 endometrioid adenocarcinoma and no myometrial invasion evaluated by magnetic resonance imaging. Eligibility criteria for chemotherapy were myometrium invasion greater than 1/2; a special type of histology, such as endometrioid adenocarcinoma grade 3, clear cell carcinoma, uterine serous papillary carcinoma, or carcinosarcoma; lymph node metastasis; or distant metastasis. Patients with residual tumor after surgery were excluded. Clinical data were collected from the medical records. All endometrial cancer specimens were obtained by surgery, and the HER2 and TOP2A status of the endometrial cancer regions was determined. Tissue microarrays were constructed as follows. We extracted a small cylindrical core from a targeted point of each paraffin-embedded
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& Volume 26, Number 2, February 2016
tissue block and re-embedded several cores into a single paraffin block, ultimately aligning them to fit on a single glass slide. In the present study, we collected from 2 points per sample, using 3-mm cores. The protocol was approved by the institutional review board of Nara Medical University as no. 960.
Immunohistochemistry Immunohistochemical staining for tissue microarray sections was performed using the polyclonal anti-2/neu (cerbB-2) antibody (1:250 dilution; Dako, Carpinteria, CA) and monoclonal topoisomerase II> antibody (1:400; Clone D10G9; Cell Signaling Technology, Beverly, MA). Immunohistochemistry was performed using formalin-fixed, paraffin-embedded tissue sections. Briefly, 2-Km-thick sections were cut using a microtome, transferred onto adhesive slides, and dried at 60-C for 30 minutes. After incubation with primary antibodies, immunohistochemistry was performed using the Leica BONDMAX systems (Mitsubishi Chemical Medicine Co, Tokyo, Japan) following the manufacturer’s instructions.
Interpretation of Immunohistochemical Staining HER2 staining was analyzed according to the guidelines of the American Society of Clinical Oncology/College of American Pathologists, as follows: a value of 0 represents no immunostaining or weak incomplete membranous staining of less than 10% of tumor cells; 1+, weak incomplete membranous staining of more than 10%; 2+, complete membrane staining that is either nonuniform or weak in intensity, but with obvious circumferential distribution of at least 10% of cells; and 3+, uniform intense membrane staining of more than 30% of invasive tumor cells.13 Scores of 2+ and 3+ were defined as HER2 positive. TOP2A staining intensity was scored as 0 to 3+ based on the percentage of positive cells: 0 (no staining), 1+ (G10%), 2+ (10%-50%), or 3+ (950%). A score of 3+ was defined as TOP2A positive. Two independent pathologists blinded to the clinical data and each other’s opinion scored the expression based on the staining intensity. Differences in opinion by the 2 pathologists were resolved by consensus.
Statistical Analysis All statistical analyses were conducted using SPSS version 17 (SPSS Inc, Chicago, IL). Probability values of less than 0.05 were considered to indicate statistical significance. Overall survival and disease-free survival between HER2 status and TOP2A status were estimated by the Kaplan-Meier method and compared using the log-rank test. The Cox proportional hazard model was used to evaluate univariate and independent multivariate associations of the clinical parameters and immunohistochemical status with overall survival and disease-free survival.
RESULTS We identified 56 patients who fulfilled the previously described criteria. Median follow-up was 49 months (range, 18Y110 months). Patient characteristics are presented in Table 1. Of the 56 patients, 36 patients (64.3%) had endometrioid adenocarcinoma (grade 1, n = 23; grade 2, n = 6; grade 3, n = 7), and 20 (35.7%) had other histologic types. Stage distribution * 2016 IGCS and ESGO
Copyright © 2016 by IGCS and ESGO. Unauthorized reproduction of this article is prohibited.
International Journal of Gynecological Cancer
& Volume 26, Number 2, February 2016 TOP2A as Marker for Endometrial Cancer
TABLE 1. Patients’ characteristics No. of patients Median age, y Median follow-up period, mo Histology
Lymphadenectomy Stage
56 61 (41Y77) 49.4 (17.5Y109.5) Endometrioid adenocarcinoma Mixed carcinoma Clear cell carcinoma Poor differentiated carcinoma Carcinosarcoma Yes No I II III IV
was as follows: 35 (62.5%) stage I, 7 (12.5%) stage II, 12 (21.4%) stage III, and 2 (3.6%) stage IV. A total of 51 patients (91.1%) underwent 6 cycles of paclitaxel (175 mg/m2) followed by carboplatin (area under the curve of 5); 5 patients (8.9%) had other taxane-platinum regimens. HER2-positive tumors were detected in 11 patients (19.6%), and TOP2Apositive tumors were detected in 7 patients (12.5%). Overall survival was not significantly different between patients with HER2-positive tumors and those with HER2-negative tumors, although disease-free survival for patients with HER2-positive tumors was significantly lower than disease-free survival for patients with HER2-negative tumors (P = 0.049; Fig. 1). In contrast, patients with TOP2A-positive tumors had
G1/G2/G3
23/6/7 9 3 2 6 49 7 35 7 12 2
significantly lower overall survival than patients with TOP2A-negative tumors (P = 0.020), and disease-free survival for patients with TOP2A-positive tumors tended to be shorter than for those with TOP2A-negative tumors (Fig. 2). Univariate analysis using the Cox proportional hazard model revealed that HER2 status, lymph node metastasis, lymph vascular space involvement, and stage were significantly associated with disease-free survival (Table 2). Multivariate analysis revealed that independent prognostic factors for disease-free survival were HER2 status and lymph vascular space involvement (P = 0.021 and 0.035, respectively; Table 2). On the other hand, univariate analysis revealed that TOP2A, lymph node metastasis, and stage
FIGURE 1. A, Kaplan-Meier estimates of overall survival by HER2 status. B, Kaplan-Meier estimates of disease-free survival by HER2 status. * 2016 IGCS and ESGO
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& Volume 26, Number 2, February 2016
FIGURE 2. A, Kaplan-Meier estimates of overall survival by TOP2A status. B, Kaplan-Meier estimates of disease-free survival by TOP2A status. were significantly associated with overall survival, whereas multivariate analysis revealed no prognostic factors for overall survival (Table 3).
DISCUSSION In the present study, TOP2A status was not detected as a prognostic factor for disease-free survival, although diseasefree survival for patients with TOP2A-positive tumors tended to be shorter than that for patients with TOP2A-negative tumors. Moreover, overall survival for patients with TOP2Apositive tumors was significantly shorter than that for patients with TOP2A-negative tumors. Based on these findings, TOP2A-positive status may predict a poor prognosis in patients with endometrial cancer receiving taxane-platinum
regimens. The metastasis-free interval in breast cancer patients with high TOP2A gene expression is significantly lower than that in patients with low expression.14 Increased TOP2A immunoexpression significantly correlated with advanced stages and tumor aggressiveness, confirming the potential of TOP2A as a prognostic biomarker for nasopharyngeal carcinoma.15 Moreover, higher expression of TOP2A protein in prostate cancer patients is a strong indicator of a poor prognosis.16 The present study revealed that TOP2A-positive status may be a predictor for poor prognosis in patients with endometrial cancer receiving taxane-platinum regimens. In other words, taxane-platinum regimens may not have a sufficient effect for TOP2A-positive endometrial cancer. Anthracycline-based neoadjuvant chemotherapy regimens
TABLE 2. Analysis of risk factors associated with disease-free survival Multivariate Variables HER2 + vs j TOP2A + vs j Lymph node metastasis + vs j Lymph vascular space involvement + vs j Myometrial invasion 91/2 vs e1/2 Histology type 2 vs type 1 Peritoneal cytology + vs j Lymphadenectomy j vs + Stages III, IV vs I, II
328
Univariate
P
95% Confidence Interval
Hazard Ratio
0.049 0.093 0.017 0.023 0.227 0.705 0.197 0.303 0.006
0.021 0.335 0.897 0.035
1.21Y10.24 0.52Y6.73 0.13Y6.17 1.08Y8.07
3.52 1.88 1.14 2.95
0.215
0.53Y16.19
2.57 * 2016 IGCS and ESGO
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& Volume 26, Number 2, February 2016 TOP2A as Marker for Endometrial Cancer
TABLE 3. Analysis of risk factors associated with overall survival Multivariate Variables
Univariate
HER2 + vs j TOP2A + vs j Lymph node metastasis + vs j Lymph vascular space involvement + vs j Myometrial invasion 91/2 vs e1/2 Histology type 2 vs type 1 Peritoneal cytology + vs j Lymphadenectomy j vs + Stages III, IV vs I, II
0.544 0.020 0.004 0.826 0.310 0.342 0.490 0.951 0.009
for breast cancer can significantly improve the pathologically complete response rate for patients with HER2 overexpression and concurrent TOP2A amplification.17 In women with HER2 geneYamplified metastatic breast cancer, overall survival in the TOP2A amplified group is significantly longer than that in the nonYTOP2A-amplified group with anthracycline-based chemotherapy.18 Thus, TOP2A status can be a predictor of anthracycline sensitivity in breast cancer. A large study should be conducted to evaluate the association between TOP2A and tumor suppression for anthracycline in endometrial cancer. In approximately 35% to 40% of patients with breast cancer with HER2 gene amplification, TOP2A is coamplified.19 This incidence of coamplification in endometrial cancer, however, is not established because there are no reports of an association between TOP2A and the effect of doxorubicin for endometrial cancer. In the present study, the frequency of both HER2 and TOP2A overexpression was 3.6% (2/56). In HER2-positive breast cancers in which TOP2A was coamplified, the rates of disease-free survival for the trastuzumab-containing regimens were indistinguishable from the rate with regimens without trastuzumab, but among HER2-positive breast cancers without TOP2A coamplification, a disease-free survival benefit with trastuzumabcontaining regimens is observed.20 On the other hand, no interaction has been detected between TOP2A expression and response to trastuzumab in patients with HER2-positive breast cancer.21 In the present study, HER2 status was detected as a significant prognostic factor for disease-free survival. Many studies report an association between HER2 and uterine serous carcinomas, with the rates of HER2 overexpression ranging from 14% to 80% and the rates of HER2 amplification ranging from 21% to 47%.22 Few studies, however, report an association between HER2 and endometrioid adenocarcinomas, and HER2 overexpression and amplification in endometrioid adenocarcinomas are reported in the range of 1% to 47% and 0% to 38%, respectively.22 Uterine serous carcinomas have a higher rate of HER2 expression compared with endometrioid carcinomas, and grade 3 endometrioid adenocarcinomas have a higher rate compared with grade 1-2 endometrioid adenocarcinomas.11 Several studies report an association between
P
95% Confidence Interval
Hazard Ratio
0.609 0.502
0.19Y16.72 0.12Y76.12
1.79 3.02
0.631
0.15Y23.04
1.85
HER2 overexpression and poor overall survival in patients with uterine serous carcinomas.12,23,24 The association between HER2 status and survival in endometrioid adenocarcinomas, however, remains uncertain. HER2 expression in the presence of amplification correlated with overall survival in a multivariate analysis.11 On the other hand, there was no significant effect of HER2 expression or amplification on survival,25 and HER2 overexpression or HER2 amplification was not associated with progression-free survival or overall survival.26 Novel HER2-based therapy in endometrial cancer has demonstrated good potential, but no significant activity was observed in the GOG-181B phase 2 trial of the single-agent trastuzumab in advanced or recurrent endometrial cancer patients.26 A multiinstitutional randomized phase 2 study (NCT01367002) is currently underway to evaluate carboplatin/paclitaxel with or without trastuzumab in patients with advanced or recurrent HER2-positive uterine serous carcinomas. Limitations of the present study include the small sample size and analysis of only immunohistochemistry. To evaluate the association between TOP2A and survival in endometrial cancer, large studies should be conducted that include analysis of TOP2A amplification in addition to immunohistochemistry. In conclusion, patients with TOP2A overexpression have a worse prognosis compared with those with TOP2A nonexpression, and TOP2A may be a useful biomarker in patients receiving adjuvant taxane-platinum regimens with moderate- to high-risk endometrial cancer. Further studies are needed to evaluate the association between TOP2A status and survival and the effects of anthracycline-containing regimens as adjuvant chemotherapy.
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