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TP53 K351N mutation-associated platinum resistance after neoadjuvant chemotherapy in patients with advanced ovarian cancer
Gynecologic Oncology 132 (2014) 752–757
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TP53 K351N mutation-associated platinum resistance after neoadjuvant chemotherapy in patients with advanced ovarian cancer Guo-nan Zhang a,c,d,1, Hong Liu a,1, Jian-ming Huang b,⁎, Ling Wang a,c, Jing-sha Zhao a,d, Chao Li a,c, Kun Mi b, Yi Zhu a,e, Jia Cheng b, Xiao Zha b a
Department of Gynaecologic Oncology, Sichuan Cancer Hospital, Chengdu, Sichuan, PR China Department of Biochemistry & Molecular Biology, Sichuan Cancer Institute, Chengdu, Sichuan, PR China Graduate School, Guangxi Medical University, Nanning, Guangxi, PR China d Graduate School, Luzhou Medical College, Luzhou, Sichuan, PR China e Department of Ultrasound, Sichuan Cancer Hospital, Chengdu, Sichuan, PR China b c
H I G H L I G H T S • TP53 K351N mutation is associated with platinum resistance after platinum-based NACT in patients with advanced ovarian cancer. • The selection of further chemotherapy after platinum-based NACT-IDS for advanced ovarian cancer should be based on TP53 K351N mutation.
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Article history: Received 14 November 2013 Accepted 16 January 2014 Available online 23 January 2014 Keywords: Ovarian cancer TP53 K351N mutation Neoadjuvant chemotherapy
a b s t r a c t Objective. TP53 K351N mutation is associated with acquired cisplatin resistance in ovarian cancer cells following exposure to cisplatin. We investigated the effect of TP53 K351N mutation on outcome in patients with epithelial ovarian cancer (EOC) who received platinum-based chemotherapy. Methods. We assessed TP53 K351N mutations by allele specific real-time PCR (AS-PCR) and DNA sequencing in tumor samples of 153 patients with stage IIIC/IV EOC. Clinicopathologic and follow-up data were collected by a retrospective chart review. Results. TP53 K351N mutations were detected in 8 (11.27%) of 71 patients who underwent neoadjuvant chemotherapy with interval debulking surgery (NACT-IDS) but not in 82 patients who underwent primary debulking surgery (PDS) (P b 0.01). In patients with relapse within 6 months, the relapse rate was 14 (19.72%) of 71 patients for NACT-IDS compared to 15 (18.29%) of 82 patients for PDS (P = 0.49), and TP53 K351N mutation was observed in 8 of NACT-IDS 14 patients (57.14% P b 0.01). In the patients retreated at first recurrence within 6 months, 7 with TP53 K351N mutation of 14 NACT-IDS patients exhibited progression of disease, compared to 2 of PDS 15 patients (50.00% vs. 13.33%, P = 0.04). The median disease-free survival (DFS) for NACT-IDS was 13.0 months compared to 15.0 months for PDS (P = 0.02). In multivariate analysis, TP53 K351N mutation is an independent factor for shorter DFS in the patients who underwent NACT-IDS (HR = 19.05; P = 0.01). Conclusions. TP53 K351N mutation may be associated with induction of platinum resistance after NACT in advanced EOC. © 2014 Elsevier Inc. All rights reserved.
Introduction Epithelial ovarian cancer (EOC) remains a leading cause of death from gynecological malignancies among the women in the world [1]. Approximately 70% of EOC patients present with advanced stage disease
⁎ Corresponding author at: Department of Biochemistry & Molecular Biology, Sichuan Cancer Institute; No. 55, Section 4, South People's Road, Chengdu 610041, Sichuan, PR China. Fax: +86 28 85420116. E-mail address: [email protected] (J. Huang). 1 Equal contributors. 0090-8258/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ygyno.2014.01.028
at the time of diagnosis and with poor response of recurrent disease to treatment [2]. Furthermore, the most effective regimen for second line chemotherapy has not yet been determined. The objective responses achieved by the second line chemotherapies are dependent on the initial progression free survival. Resistance to platinum chemotherapy, either de novo or acquired, presents a substantial obstacle to attempt to improve the prognosis of patients with advanced EOC. Initially responsive, 20–30% of patient will have recurrence within 6 months of completion of primary platinum therapy [3,4]. Currently, around 50% of the patients with stage IIIC/IV ovarian cancer are selected for neoadjuvant chemotherapy with interval debulking surgery (NACT-IDS) as a
G. Zhang et al. / Gynecologic Oncology 132 (2014) 752–757
treatment option [5–7]. Recently, the findings from several trials reported that patients with stages IIIC/IV EOC randomized to NACT-IDS had the same survival as patients undergoing primary debulking surgery (PDS) followed by chemotherapy and that NACT-IDS was not inferior to PDS [8–10]. Despite recent data appear to support a paradigm shift toward NACT-IDS for a subset of patients with stages IIIC/IV EOC, it remains controversial whether the better outcome is due to NACT-IDS followed by platinum-based chemotherapy [11,12]. However, a recent data reported that in patients with stages IIIC/IV EOC who had a relapse within 6 months and are retreated with platinum-based chemotherapy, NACTIDS contributed to an increased risk of platinum resistance [13]. But the causes of further relapse with platinum-resistant disease after NACT remain unclear. Despite increasing efforts in terms of more radical surgery and more efficient systemic treatment, survival of women with advanced EOC has changed little since platinum-based treatment was introduced over 30 years ago. Accordingly, it is important for the management of advanced EOC to elucidate the mechanisms of chemoresistance and to get over the resistance. Both intrinsic and acquired resistance during treatment results from the numerous genetic and epigenetic changes occurring in cancer cells [14]. The success of platinum-based chemotherapy is limited due to its pre-existed or acquired drug resistance. It is well established that more than 50% of advanced ovarian cancers contain TP53 mutations that are mostly missense [15]. Although TP53 mutation status was not predictive of survival (disease free and overall) or of chemoresistance [16–20], the TP53 missense mutation-mediated cisplatin resistance may be associated with reduced transactivation and expression of BAX, an apoptosis-inducing gene, in ovarian carcinoma cell systems [19]. These studies reported that TP53 mutations in ovarian cancers were mostly involved in exons of DNA binding domains (17) but not involved in the tetramerization domain of TP53 that is responsible for its nuclear export signal (NES) and translocation to mitochondria (Supplementary Fig. 1). A missense mutation of G to T transversion at codon 351 (Lys 351 to Asn, K351N) in the tetramerization domain (TD) in exon 10 of TP53 in cisplatin-resistant subpopulation of human EOC cell line (A2780CIS) has been found to be related to acquired resistance to cisplatin. A2780CIS cell line has been developed by chronic exposure of the parent cisplatinsensitive A2780 cell line to cisplatin [21–23]. An increased ability to repair DNA damage as well as cytogenetic abnormalities has been observed in A2780CIS cell line [24]. TP53 K351N mutation results in a combined loss of tetramerization and nuclear export or translocation to mitochondria of TP53 protein which mediates the activation of BAX, leading to defects in TP53 apoptosis activity [19,25,26]. This defect in TP53 apoptosis activity is critical for the development and maintenance of resistance to platinum in EOC cells. If tumors of patients with stages IIIC/IV EOC after platinumbased NACT-IDS are found to have a TP53 K351N mutation, their tumors likely become resistant to further platinum-based chemotherapy. The objective of this study is to investigate whether TP53 K351N mutations emerge in tumors of patients with stages IIIC/IV EOC after platinum-based NACT and to assess the effect of TP53 K351N mutation on outcome of treatment in a clinical setting. Patients and methods
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NACT-IDS (2–7 cycles, 3.89 ± 1.60, of platinum-based chemotherapy before IDS, and 82 (53.59%) PDS in the Sichuan Cancer Hospital between April 1999 to August 2009, were available for analysis of TP53 K351N mutation. All the patients with EOC enrolled in the study were histopathologically confirmed to be suffering from serous ovarian cancer by a tissue biopsy before initiation of therapy. The surgical procedures were performed by the Department of Gynecologic Oncology, with an intent to achieve optimal cytoreduction. Optimal cytoreduction was defined as less than or equal to 1-cm maximal diameter of the largest residual tumor nodule at the completion of the primary operation. Paclitaxel/platinum-based chemotherapy was largely used in this study. Clinicopathologic and follow-up data of patients were collected by a retrospective chart review. All patients, enrolled in this study, had signed informed consent, approved by our Internal Ethical Committee.
Evaluation of response to treatment Evaluation of response to treatment was performed in all patients, based on revised RECIST criteria (version 1.1) [27]. Response to therapy was determined by disease-free survival (DFS) in all patients, which was defined as the period from PDS or initial diagnosis to recurrence.
Detection of the K351N mutation of TP53 by AS-PCR assay Genomic DNA was extracted from cell lines and tumor specimens using High pure PCR Template Preparation Kit (Roche,Germany) and QIAamp DNA FFPE Tissue Kit (Qiagen,U.K.), respectively according to the manufacturer's protocols and stored at −20 °C until analysis. The K351N mutant (AAG → AAT) in TP53 exon 10 (GenBank: M13120.1) was prepared by PCR with site-directed mutagenic primers as a positive control, and the wild-type allele in TP53 exon 10 was used as a reference. All primers and fluorogenic MGB probes were designed (Supplementary Table 1) using Primer Express 3.0 software (Applied Biosystems) and synthesized by GeneCore Bio Technologies Corporation, Shanghai, China and DNAchem Corporation, Beijing, China, respectively. Thermal cycling procedures for AS-PCR were performed routinely.
Interpretation of AS-PCR results The CtHEX for the specific reaction reflects the amount of TP53 K351N mutation within the sample, while the CtFAM for the reference reaction reflects the total amount of TP53 allelic template in the sample. ΔCt cut-off value was measured using a CtHEX value of 10−9 dilution of positive template, mixed into normal DNA template, subtracted by the CtFAM value of allelic reference, and the mean ΔCt value of less than ΔCt cut-off value for each sample was defined as TP53 K351N positive mutation. When the ΔCt measure exceeds ΔCt cut-off values for the mutant reactions, the assay reports no mutation detected.
Tumor cell lines Statistical analysis Human EOC A2780 cell line (ECACC catalog code: 93112519) and its cisplatin-resistant sub-line A2780CIS (ECACC catalog code: 93112517) were purchased from the Committee on Type Culture Collection of Chinese Academy of Sciences (CTCCCAS, Shanghai, China) and were routinely passaged in RPMI-1640 medium (GIBCO, Invitrogen). Patients and tumor samples For the present study, paraffin-embedded tumor samples of 153 patients with FIGO stage IIIC/IV EOC, consisting of 71 (46.41%) underwent
This study was performed in accordance with the criteria of reporting recommendations for tumour marker prognostic studies (REMARK) [28]. All statistical calculations were performed with the use of SPSS software (version 17.0). The positive rate of TP53 K351N mutation in patients with EOC was calculated by Clopper–Pearson with adjusted coefficient k (0.5). The relationship between TP53 K351N mutation status and DFS was analyzed with the Kaplan–Meier method with a log-rank (Mantel–Cox) test. Two-sided P value of less than 0.05 was considered to indicate statistical significance.
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Results
TP53 K351N mutation and survival analysis
Specificity and sensitivity of AS-PCR assay
The median DFS for NACT-IDS was 13.0 months (95% CI, 10.52 to 15.48 months) compared to 15.0 months (95%CI, 13.50 to 16.50 months) for PDS (Fig. 1A). The NACT-IDS patients exhibited significantly shorter DFS as compared with PDS patients (P = 0.02). When the 63 (88.7%, 63/71) patients who don't have TP 53 K351N mutation in the NACT-IDS group were compared to the 82 patients who underwent PDS, the median DFS for NACT-IDS and for PDS were 14 months (95% CI, 12.89 to 15.11 months) and 15 months (95% CI, 13.50 to 16.50 months), respectively (P = 0.10) (Fig. 1B). In multivariate analysis, TP53 K351N mutation is an independent factor for shorter DFS in the patients who underwent NACT-IDS (HR = 19.05; P = 0.01) (Fig. 1C and Table 1).
We tested the specificity and sensitivity of the allele-specific PCR assay for detecting TP53 K351N mutation in the positive control incorporated into the DNA template with wild-type allele. Our results showed the TP53 K351N missense mutation (AAG → AAT) of exon 10 in the positive control on DNA sequencing, and no overlap between the allele-specific primers and probes, and the signals of nonspecific hybridization and amplification in this AS-PCR assay (Supplementary Fig. 2A). A typical standard curve amplification plot and linear regression analysis (10−4 to 10−9 dilution of positive control) yielded a correlation coefficient of 0.992 and a y-intercept value of 24.49 and a slope of −3.783 which closely approximated the theoretical maximum amplification efficiency of 100% (−3.32 slope) (Supplementary Fig. 2A). The mean ΔCt value (4.84 ± 0.46, range 4.38–5.30) obtained by the subtraction of a CtHEX value of 10−9 from the CtFAM value of allelic reference was defined as the ΔCt cut-off value. For TP53 K351N, the mean ΔCt cut-off value of all genotypically mutant specimens for each assay ≤ 5 or N 5 was interpreted as mutation positive or mutation negative, respectively. Detection of TP53 K351N mutation in EOC cell lines This test is designed to detect platinum resistance-associated types of TP53 K351N mutation. To validate the results of this AS-PCR assay, we tested the suitability of this AS-PCR assay for detecting TP53 K351N mutation in cisplatin-resistant A2780CIS and cisplatin-sensitive A2780 cells previously identified as having the TP53 K351N mutation and as having wild-type alleles, respectively. This AS-PCR assay and sequencing identified the same TP53 K351N mutation in A2780CIS cells, whereas A2780 were confirmed as being negative, yielding a sensitivity of 96% and a specificity of 100%. This method could be used as a reliable test for TP53 K351N mutation. Identification of TP53 K351N mutations in tumors Using the AS-PCR assay, we analyzed paraffin-embedded tumors of 153 patients with stage IIIC/IV EOC previously treated with NACT-IDS and PDS followed by chemotherapy. In three independent runs, we identified TP53 K351N mutations (mean ΔCt N cut-off value) in 8 (11.27%, 95% CI, 4.98%–20.98%) of 71 NACT-IDS patients but not in 82 PDS patients. Platinum-based NACT exhibited a higher rate of TP53 K351N mutation, but the number of cycles did not significantly influence the rate of TP53 K351N mutation (P = 0.10). The accordant results between sequencing and AS-PCR assay were quantitatively and qualitatively verified (Supplementary Fig. 2B–D). TP53 K351N mutation and response to therapy Response to therapy was analyzed in the 153 patients who received platinum-based chemotherapy. The overall response rate to platinumbased chemotherapy in the NACT-IDS group was 80.28%, compared to an overall response rate of 82.92% in the PDS group (P = 0.42); 14 (19.72%) of 71 NACT-IDS patients relapsed within 6 months after completion of platinum-based chemotherapy, compared to 15 (18.29%) of 82 PDS patients (P = 0.49). However, in patients with relapse within 6 months after completion of NACT-IDS and PDS followed by chemotherapy, TP53 K351N mutation was observed in 8 of 14 NACT-IDS patients, and the rate of TP53 K351N mutation was significantly higher in NACT-IDS compared to PDS patients (57.14% vs. 0%, P b 0.01). In the 29 patients retreated with platinum-based chemotherapy at first recurrence within 6 months, 7 (50.00%), with TP 53 K351N mutation, of 14 patients initially treated with NACT exhibited a higher rate of progression while on therapy, compared to 2 (13.33%) of 15 patients treated with PDS (P = 0.04).
Discussion The development of chemoresistance prevents the efficient treatment of recurrent and progressive ovarian cancer. Despite advances in surgery and therapy, overall prognosis remains relatively poor, and relapse within 6 months and progression to chemotherapy resistance are frequently seen in many patients with advanced EOC [29]. It has been evidenced that EOC patients retreated with platinum-based chemotherapy at first recurrence after initial NACT-IDS exhibited a higher rate of progression and had platinum resistant disease at interval to second recurrence less than 6 months [13,30]. A better understanding of what it is about ovarian cancer that allows it to be platinum resistant to further therapy after an initial platinum-based NACT is needed for better treatment strategies that could circumvent platinum resistance. Both intrinsic and acquired resistance result from the numerous genetic and epigenetic changes occurring in cancer cells. Resistance to chemotherapy is believed to occur by two mechanisms: either selection of a population of tumor cells inherently resistant to the agents employed in treatment or the induction of resistance of these tumor cells by the chemotherapeutics themselves [31]. Some studies have postulated that a small subpopulation of intrinsic resistant cancer cells can continue to grow after treatment and will repopulate the tumor bulk, owing to the removal of competing or sensitive cells, whereas other studies have proposed that genetic changes or mutations may confer acquired cisplatin resistance during cancer chemotherapy [32]. However, whether resistance mutations have any selective advantage in the absence of therapy, or whether they are merely low-frequency events until the application of chemotherapy needs to be further investigated. Various lines of evidence indicate that cancer-associated TP53 mutations can contribute actively to tumor progression, increased resistance to platinum-based chemotherapy and shortened survival in ovarian cancer [18,19]. However, a review of the extensive literature on TP53 in ovarian cancer does not support a consistent relationship between TP53 mutation and response to therapy or survival in multivariate analysis [18,20]. This may reflect the complexity that may be an indication that mutations in the coding sequence represent only a subset of the functional TP53 alterations. The TP53 K351N mutation is believed to be linked to acquisition of platinum-resistance in ovarian cancer A2780 cells following cisplatin treatment. Mutations of the nuclear export signal (NES, Met340– Lys351) in the TD prevent TP53 nuclear export and tetramerization that are indispensable for TP53/BAX/caspases signaling-mediated cellular apoptosis [19,25]. The TP53 K351N mutant greatly reduced the ability to interact with BAK and BAK oligomerization, then leading to resistance to cisplatin-induced apoptosis [25,26]. It has been shown that in ovarian cancers, the resistance to cisplatin is due to the loss of mismatch repair (MMR) of DNA capable of increasing mismatch mutation rates at the specific genes, including TP53 gene, that control response to chemotherapy [33,34]. As cisplatin is itself a mutagen, it may aid the process of creating and developing a mutant
G. Zhang et al. / Gynecologic Oncology 132 (2014) 752–757 Fig. 1. Correlation between the TP53 K351N mutations in tumor specimen and shortened disease-free survival. (A) DFS for NACT-IDS vs. PDS. (B) DFS for NACT-IDS excluded the TP53 K351N mutant patients vs. PDS. (C) In NACT-IDS patients, the TP53 K351N mutation was associated with shortened DFS.
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Table 1 Cox regression analysis for recurrence within six months after NACT-IDS (N = 71). Disease-free survival
Univariate
Variables
HR(95%CI)
P value
Multivariate
Age (≥55 years vs b55 years) Histological grade (poor vs well/moderate) Malignant cells in ascites (yes vs no) Lymph node metastasis (yes vs no) Liver or lung metastasis (yes vs no) Residual tumor (N1 vs b1) NACT (N3 vs ≤3) K351N (yes vs no)
1.84 (0.62–5.50) 2.40 (0.75–7.65) 1.71 (0.38–7.67) 0.64 (0.18–2.28) 0.23 (0.08–0.70) 1.06 (0.37–3.06) 2.01 (0.67–5.99) 13.96 (4.82–40.47)
0.27 0.14 0.48 0.49 0.01 0.91 0.21 b0.01
HR(95%CI)
P value
1.47 (0.17–12.59)
0.73
19.05(2.37–152.77)
0.01
Abbreviations: DFS = disease-free survival; HR = hazard ratio; CI = confidence interval. Bold values indicate P b 0.05.
cell that is itself resistant to the treatment process and is a possible origin of platinum-resistant ovarian cancer cells following exposure to cisplatin [35–38]. Therefore, we wonder whether the platinum resistance disease in advanced EOC after platinum-based NACT-IDS arises owing to TP53 K351N mutations that develop during or following the first treatment or that pre-exist within the tumour bulk at the time of first treatment in a clinical setting. In this study, we showed for the first time that occurrence of TP53 K351N mutation after platinum-based NACT correlates with the platinum resistance of EOC in a clinical setting; We found that after initial treatment, when multivariate logistic regression was used to control for factors independently associated with recurrence within 6 months, NACT-IDS did not appear significantly to influence the recurrence rates within 6 months compared to PDS (P = 0.49). However, when response to treatment was evaluated in the 29 patients retreated with platinum-based chemotherapy at first recurrence within 6 months, half of the patients initially treated with NACT, having TP53 K351N mutation, exhibited an extremely poor response to the second-line platinum-based chemotherapy, with interval to second recurrence less than 6 months (P = 0.04), as reported in Ref.13. In fact, we also found that TP53 K351N mutations occurred in more than half of the NACTIDS patients relapsed within 6 months, suggesting that loss of MMR results in rapid evolution of platinum resistance during sequential rounds of drug exposure [31,34]. Moreover, when excluding the TP53 K351N mutation-positive patients, the difference in DFS between NACT-IDS and PDS was lost (P = 0.10) (Fig. 1B). Actually, in multivariate analysis, the TP53 K351N mutation as an independent factor significantly influenced DFS of EOC patients who received NACT (P b 0.001) (Fig. 1C and Table 1). Our data support the emergence of resistance mutations following treatment in some cases of relapsed ovarian carcinoma, since platinum-resistance disease at second relapse in those patients who received NACT-IDS occurs more frequently than does in those patients who received PDS, suggesting that NACT administration, when tumor burden is large and heterogeneous, may induce TP53 K351N mutant clones resulting in resistance of these tumor cells to platinum therapy through spontaneously mutant selection or cellular selfdefense mechanism resulting from genetic changes [39,40]. Our findings may explain at least to some extent that TP53 K351N mutation, which is distinct from mutations in other functional domains of the TP53 gene, plays a crucial role in conferring platinum resistance to second-line platinum-based chemotherapy after NACT in advanced EOC. The collection of sequential samples from patients before and after platinum-based chemotherapy will be critical for detection of small populations of TP53 K351N mutant subclones as even with nextgeneration sequencing. Based on TP53 K351N mutation, the selection of non-platinum-based chemotherapy for further treatment after platinum-based NACT-IDS would improve the outcome of NACT-IDS followed by chemotherapy. In conclusion, our study indicates that the TP53 K351N mutation may be associated with induction of platinum resistance after platinum-based
NACT in advanced EOC. As we would expect, a prospective clinical study is necessary to get reliable data on the TP53 K351N mutation-associated platinum resistance in ovarian cancer cells after platinum-based NACT. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.ygyno.2014.01.028.
Conflict of interest statement The authors have no conflicts of interest to declare.
References [1] Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA Cancer J Clin 2013;63(1): 11–30. [2] Vasey PA, Herrstedt J, Jelic S, Force EGT. ESMO Minimum Clinical Recommendations for diagnosis, treatment and follow-up of epithelial ovarian carcinoma. Ann Oncol 2005;16(Suppl. 1):i13–5. [3] Perez RP, Hamilton TC, Ozols RF, Young RC. Mechanisms and modulation of resistance to chemotherapy in ovarian cancer. Cancer 1993;71(4 Suppl.):1571–80. [4] Kelland L. The resurgence of platinum-based cancer chemotherapy. Nat Rev Cancer 2007;7(8):573–84. [5] Cornelis S, Van Calster B, Amant F, Leunen K, van der Zee AG, Vergote I. Role of neoadjuvant chemotherapy in the management of stage IIIC-IV ovarian cancer: survey results from the members of the European Society of Gynecological Oncology. Int J Gynecol Cancer 2012;22(3):407–16. [6] Worley Jr MJ, Guseh SH, Rauh-Hain JA, Williams KA, Muto MG, Feltmate CM, et al. Does neoadjuvant chemotherapy decrease the risk of hospital readmission following debulking surgery? Gynecol Oncol 2013;129(1):69–73. [7] Morrison J, Haldar K, Kehoe S, Lawrie TA. Chemotherapy versus surgery for initial treatment in advanced ovarian epithelial cancer. Cochrane Database Syst Rev 2012;8:CD005343. [8] Vergote I, Trope CG, Amant F, Kristensen GB, Ehlen T, Johnson N, et al. Neoadjuvant chemotherapy or primary surgery in stage IIIC or IV ovarian cancer. N Engl J Med 2010;363(10):943–53. [9] Vergote I, Trope CG, Amant F, Ehlen T, Reed NS, Casado A. Neoadjuvant chemotherapy is the better treatment option in some patients with stage IIIc to IV ovarian cancer. J Clin Oncol 2011;29(31):4076–8. [10] Vergote I, Amant F, Kristensen G, Ehlen T, Reed NS, Casado A. Primary surgery or neoadjuvant chemotherapy followed by interval debulking surgery in advanced ovarian cancer. Eur J Cancer 2011;47(Suppl. 3):S88–92. [11] Chi DS, Musa F, Dao F, Zivanovic O, Sonoda Y, Leitao MM, et al. An analysis of patients with bulky advanced stage ovarian, tubal, and peritoneal carcinoma treated with primary debulking surgery (PDS) during an identical time period as the randomized EORTC-NCIC trial of PDS vs neoadjuvant chemotherapy (NACT). Gynecol Oncol 2012;124(1):10–4. [12] Jelovac D, Armstrong DK. Recent progress in the diagnosis and treatment of ovarian cancer. CA Cancer J Clin 2011;61(3):183–203. [13] Rauh-Hain JA, Nitschmann CC, Worley Jr MJ, Bradford LS, Berkowitz RS, Schorge JO, et al. Platinum resistance after neoadjuvant chemotherapy compared to primary surgery in patients with advanced epithelial ovarian carcinoma. Gynecol Oncol 2013;129(1):63–8. [14] Agarwal R, Kaye SB. Ovarian cancer: strategies for overcoming resistance to chemotherapy. Nat Rev Cancer 2003;3(7):502–16. [15] Skilling JS, Squatrito RC, Connor JP, Niemann T, Buller RE. p53 gene mutation analysis and antisense-mediated growth inhibition of human ovarian carcinoma cell lines. Gynecol Oncol 1996;60(1):72–80. [16] Laframboise S, Chapman W, McLaughlin J, Andrulis IL. p53 mutations in epithelial ovarian cancers: possible role in predicting chemoresistance. Cancer J 2000;6(5): 302–8. [17] Gadducci A, Di Cristofano C, Zavaglia M, Giusti L, Menicagli M, Cosio S, et al. P53 gene status in patients with advanced serous epithelial ovarian cancer in relation to response to paclitaxel- plus platinum-based chemotherapy and long-term clinical outcome. Anticancer Res 2006;26(1B):687–93.
G. Zhang et al. / Gynecologic Oncology 132 (2014) 752–757 [18] Reles A, Wen WH, Schmider A, Gee C, Runnebaum IB, Kilian U, et al. Correlation of p53 mutations with resistance to platinum-based chemotherapy and shortened survival in ovarian cancer. Clin Cancer Res 2001;7(10):2984–97. [19] Perego P, Giarola M, Righetti SC, Supino R, Caserini C, Delia D, et al. Association between cisplatin resistance and mutation of p53 gene and reduced bax expression in ovarian carcinoma cell systems. Cancer Res 1996;56(3):556–62. [20] Ahmed AA, Etemadmoghadam D, Temple J, Lynch AG, Riad M, Sharma R, et al. Driver mutations in TP53 are ubiquitous in high grade serous carcinoma of the ovary. J Pathol 2010;221(1):49–56. [21] Muscolini M, Montagni E, Caristi S, Nomura T, Kamada R, Di Agostino S, et al. Characterization of a new cancer-associated mutant of p53 with a missense mutation (K351N) in the tetramerization domain. Cell Cycle 2009;8(20):3396–405. [22] Turanek J, Kasna A, Zaluska D, Neca J, Kvardova V, Knotigova P, et al. New platinum(IV) complex with adamantylamine ligand as a promising anti-cancer drug: comparison of in vitro cytotoxic potential towards A2780/cisR cisplatinresistant cell line within homologous series of platinum(IV) complexes. Anticancer Drugs 2004;15(5):537–43. [23] Behrens BC, Hamilton TC, Masuda H, Grotzinger KR, Whang-Peng J, Louie KG, et al. Characterization of a cis-diamminedichloroplatinum(II)-resistant human ovarian cancer cell line and its use in evaluation of platinum analogues. Cancer Res 1987;47(2):414–8. [24] Masuda H, Ozols RF, Lai GM, Fojo A, Rothenberg M, Hamilton TC. Increased DNA repair as a mechanism of acquired resistance to cis-diamminedichloroplatinum (II) in human ovarian cancer cell lines. Cancer Res 1988;48(20):5713–6. [25] Muscolini M, Montagni E, Palermo V, Di Agostino S, Gu W, Abdelmoula-Souissi S, et al. The cancer-associated K351N mutation affects the ubiquitination and the translocation to mitochondria of p53 protein. J Biol Chem 2011;286(46):39693–702. [26] Kamada R, Nomura T, Anderson CW, Sakaguchi K. Cancer-associated p53 tetramerization domain mutants: quantitative analysis reveals a low threshold for tumor suppressor inactivation. J Biol Chem 2011;286(1):252–8. [27] Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R, et al. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer 2009;45(2):228–47. [28] McShane LM, Altman DG, Sauerbrei W, Taube SE, Gion M, Clark GM. Statistics Subcommittee of the NCIEWGoCD: reporting recommendations for tumor marker prognostic studies. J Clin Oncol 2005;23(36):9067–72.
757
[29] Vergote I, Rustin GJ, Eisenhauer EA, Kristensen GB, Pujade-Lauraine E, Parmar MK, et al. Re: new guidelines to evaluate the response to treatment in solid tumors [ovarian cancer]. Gynecologic Cancer Intergroup. J Natl Cancer Inst 2000;92(18): 1534–5. [30] Bristow RE, Eisenhauer EL, Santillan A, Chi DS. Delaying the primary surgical effort for advanced ovarian cancer: a systematic review of neoadjuvant chemotherapy and interval cytoreduction. Gynecol Oncol 2007;104(2):480–90. [31] Cooke SL, Brenton JD. Evolution of platinum resistance in high-grade serous ovarian cancer. Lancet Oncol 2011;12(12):1169–74. [32] Galluzzi L, Senovilla L, Vitale I, Michels J, Martins I, Kepp O, et al. Molecular mechanisms of cisplatin resistance. Oncogene 2012;31(15):1869–83. [33] Branch P, Masson M, Aquilina G, Bignami M, Karran P. Spontaneous development of drug resistance: mismatch repair and p53 defects in resistance to cisplatin in human tumor cells. Oncogene 2000;19:3138–45. [34] Lin X, Howell SB. DNA mismatch repair and p53 function are major determinants of the rate of development of cisplatin resistance. Mol Cancer Ther 2006;5(5): 1239–47. [35] Xiao X, Melton DW, Gourley C. Mismatch repair deficiency in ovarian cancer— molecular characteristics and clinical implications. Gynecol Oncol 2014;132(2): 506–12. [36] Aebi S, Kurdi-Haidar B, Gordon R, Cenni B, Zheng H, Fink D, et al. Loss of DNA mismatch repair in acquired resistance to cisplatin. Cancer Res 1996;56(13): 3087–90. [37] de las Alas MM, Aebi S, Fink D, Howell SB, Los G. Loss of DNA mismatch repair: effects on the rate of mutation to drug resistance. J Natl Cancer Inst 1997;89(20): 1537–41. [38] Gercel-Taylor C, Scobee JJ, Taylor DD. Effect of chemotherapy on the mutation frequency of ovarian cancer cells at the HPRT locus. Anticancer Res 2005;25(3B): 2113–7. [39] Righetti SC, Perego P, Corna E, Pierotti MA, Zunino F. Emergence of p53 mutant cisplatin-resistant ovarian carcinoma cells following drug exposure: spontaneously mutant selection. Cell Growth Differ 1999;10(7):473–8. [40] Shen DW, Pouliot LM, Hall MD, Gottesman MM. Cisplatin resistance: a cellular selfdefense mechanism resulting from multiple epigenetic and genetic changes. Pharmacol Rev 2012;64(3):706–21.
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TP53 K351N mutation-associated platinum resistance after neoadjuvant chemotherapy in patients with advanced ovarian cancer Guo-nan Zhang a,c,d,1, Hong Liu a,1, Jian-ming Huang b,⁎, Ling Wang a,c, Jing-sha Zhao a,d, Chao Li a,c, Kun Mi b, Yi Zhu a,e, Jia Cheng b, Xiao Zha b a
Department of Gynaecologic Oncology, Sichuan Cancer Hospital, Chengdu, Sichuan, PR China Department of Biochemistry & Molecular Biology, Sichuan Cancer Institute, Chengdu, Sichuan, PR China Graduate School, Guangxi Medical University, Nanning, Guangxi, PR China d Graduate School, Luzhou Medical College, Luzhou, Sichuan, PR China e Department of Ultrasound, Sichuan Cancer Hospital, Chengdu, Sichuan, PR China b c
H I G H L I G H T S • TP53 K351N mutation is associated with platinum resistance after platinum-based NACT in patients with advanced ovarian cancer. • The selection of further chemotherapy after platinum-based NACT-IDS for advanced ovarian cancer should be based on TP53 K351N mutation.
a r t i c l e
i n f o
Article history: Received 14 November 2013 Accepted 16 January 2014 Available online 23 January 2014 Keywords: Ovarian cancer TP53 K351N mutation Neoadjuvant chemotherapy
a b s t r a c t Objective. TP53 K351N mutation is associated with acquired cisplatin resistance in ovarian cancer cells following exposure to cisplatin. We investigated the effect of TP53 K351N mutation on outcome in patients with epithelial ovarian cancer (EOC) who received platinum-based chemotherapy. Methods. We assessed TP53 K351N mutations by allele specific real-time PCR (AS-PCR) and DNA sequencing in tumor samples of 153 patients with stage IIIC/IV EOC. Clinicopathologic and follow-up data were collected by a retrospective chart review. Results. TP53 K351N mutations were detected in 8 (11.27%) of 71 patients who underwent neoadjuvant chemotherapy with interval debulking surgery (NACT-IDS) but not in 82 patients who underwent primary debulking surgery (PDS) (P b 0.01). In patients with relapse within 6 months, the relapse rate was 14 (19.72%) of 71 patients for NACT-IDS compared to 15 (18.29%) of 82 patients for PDS (P = 0.49), and TP53 K351N mutation was observed in 8 of NACT-IDS 14 patients (57.14% P b 0.01). In the patients retreated at first recurrence within 6 months, 7 with TP53 K351N mutation of 14 NACT-IDS patients exhibited progression of disease, compared to 2 of PDS 15 patients (50.00% vs. 13.33%, P = 0.04). The median disease-free survival (DFS) for NACT-IDS was 13.0 months compared to 15.0 months for PDS (P = 0.02). In multivariate analysis, TP53 K351N mutation is an independent factor for shorter DFS in the patients who underwent NACT-IDS (HR = 19.05; P = 0.01). Conclusions. TP53 K351N mutation may be associated with induction of platinum resistance after NACT in advanced EOC. © 2014 Elsevier Inc. All rights reserved.
Introduction Epithelial ovarian cancer (EOC) remains a leading cause of death from gynecological malignancies among the women in the world [1]. Approximately 70% of EOC patients present with advanced stage disease
⁎ Corresponding author at: Department of Biochemistry & Molecular Biology, Sichuan Cancer Institute; No. 55, Section 4, South People's Road, Chengdu 610041, Sichuan, PR China. Fax: +86 28 85420116. E-mail address: [email protected] (J. Huang). 1 Equal contributors. 0090-8258/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ygyno.2014.01.028
at the time of diagnosis and with poor response of recurrent disease to treatment [2]. Furthermore, the most effective regimen for second line chemotherapy has not yet been determined. The objective responses achieved by the second line chemotherapies are dependent on the initial progression free survival. Resistance to platinum chemotherapy, either de novo or acquired, presents a substantial obstacle to attempt to improve the prognosis of patients with advanced EOC. Initially responsive, 20–30% of patient will have recurrence within 6 months of completion of primary platinum therapy [3,4]. Currently, around 50% of the patients with stage IIIC/IV ovarian cancer are selected for neoadjuvant chemotherapy with interval debulking surgery (NACT-IDS) as a
G. Zhang et al. / Gynecologic Oncology 132 (2014) 752–757
treatment option [5–7]. Recently, the findings from several trials reported that patients with stages IIIC/IV EOC randomized to NACT-IDS had the same survival as patients undergoing primary debulking surgery (PDS) followed by chemotherapy and that NACT-IDS was not inferior to PDS [8–10]. Despite recent data appear to support a paradigm shift toward NACT-IDS for a subset of patients with stages IIIC/IV EOC, it remains controversial whether the better outcome is due to NACT-IDS followed by platinum-based chemotherapy [11,12]. However, a recent data reported that in patients with stages IIIC/IV EOC who had a relapse within 6 months and are retreated with platinum-based chemotherapy, NACTIDS contributed to an increased risk of platinum resistance [13]. But the causes of further relapse with platinum-resistant disease after NACT remain unclear. Despite increasing efforts in terms of more radical surgery and more efficient systemic treatment, survival of women with advanced EOC has changed little since platinum-based treatment was introduced over 30 years ago. Accordingly, it is important for the management of advanced EOC to elucidate the mechanisms of chemoresistance and to get over the resistance. Both intrinsic and acquired resistance during treatment results from the numerous genetic and epigenetic changes occurring in cancer cells [14]. The success of platinum-based chemotherapy is limited due to its pre-existed or acquired drug resistance. It is well established that more than 50% of advanced ovarian cancers contain TP53 mutations that are mostly missense [15]. Although TP53 mutation status was not predictive of survival (disease free and overall) or of chemoresistance [16–20], the TP53 missense mutation-mediated cisplatin resistance may be associated with reduced transactivation and expression of BAX, an apoptosis-inducing gene, in ovarian carcinoma cell systems [19]. These studies reported that TP53 mutations in ovarian cancers were mostly involved in exons of DNA binding domains (17) but not involved in the tetramerization domain of TP53 that is responsible for its nuclear export signal (NES) and translocation to mitochondria (Supplementary Fig. 1). A missense mutation of G to T transversion at codon 351 (Lys 351 to Asn, K351N) in the tetramerization domain (TD) in exon 10 of TP53 in cisplatin-resistant subpopulation of human EOC cell line (A2780CIS) has been found to be related to acquired resistance to cisplatin. A2780CIS cell line has been developed by chronic exposure of the parent cisplatinsensitive A2780 cell line to cisplatin [21–23]. An increased ability to repair DNA damage as well as cytogenetic abnormalities has been observed in A2780CIS cell line [24]. TP53 K351N mutation results in a combined loss of tetramerization and nuclear export or translocation to mitochondria of TP53 protein which mediates the activation of BAX, leading to defects in TP53 apoptosis activity [19,25,26]. This defect in TP53 apoptosis activity is critical for the development and maintenance of resistance to platinum in EOC cells. If tumors of patients with stages IIIC/IV EOC after platinumbased NACT-IDS are found to have a TP53 K351N mutation, their tumors likely become resistant to further platinum-based chemotherapy. The objective of this study is to investigate whether TP53 K351N mutations emerge in tumors of patients with stages IIIC/IV EOC after platinum-based NACT and to assess the effect of TP53 K351N mutation on outcome of treatment in a clinical setting. Patients and methods
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NACT-IDS (2–7 cycles, 3.89 ± 1.60, of platinum-based chemotherapy before IDS, and 82 (53.59%) PDS in the Sichuan Cancer Hospital between April 1999 to August 2009, were available for analysis of TP53 K351N mutation. All the patients with EOC enrolled in the study were histopathologically confirmed to be suffering from serous ovarian cancer by a tissue biopsy before initiation of therapy. The surgical procedures were performed by the Department of Gynecologic Oncology, with an intent to achieve optimal cytoreduction. Optimal cytoreduction was defined as less than or equal to 1-cm maximal diameter of the largest residual tumor nodule at the completion of the primary operation. Paclitaxel/platinum-based chemotherapy was largely used in this study. Clinicopathologic and follow-up data of patients were collected by a retrospective chart review. All patients, enrolled in this study, had signed informed consent, approved by our Internal Ethical Committee.
Evaluation of response to treatment Evaluation of response to treatment was performed in all patients, based on revised RECIST criteria (version 1.1) [27]. Response to therapy was determined by disease-free survival (DFS) in all patients, which was defined as the period from PDS or initial diagnosis to recurrence.
Detection of the K351N mutation of TP53 by AS-PCR assay Genomic DNA was extracted from cell lines and tumor specimens using High pure PCR Template Preparation Kit (Roche,Germany) and QIAamp DNA FFPE Tissue Kit (Qiagen,U.K.), respectively according to the manufacturer's protocols and stored at −20 °C until analysis. The K351N mutant (AAG → AAT) in TP53 exon 10 (GenBank: M13120.1) was prepared by PCR with site-directed mutagenic primers as a positive control, and the wild-type allele in TP53 exon 10 was used as a reference. All primers and fluorogenic MGB probes were designed (Supplementary Table 1) using Primer Express 3.0 software (Applied Biosystems) and synthesized by GeneCore Bio Technologies Corporation, Shanghai, China and DNAchem Corporation, Beijing, China, respectively. Thermal cycling procedures for AS-PCR were performed routinely.
Interpretation of AS-PCR results The CtHEX for the specific reaction reflects the amount of TP53 K351N mutation within the sample, while the CtFAM for the reference reaction reflects the total amount of TP53 allelic template in the sample. ΔCt cut-off value was measured using a CtHEX value of 10−9 dilution of positive template, mixed into normal DNA template, subtracted by the CtFAM value of allelic reference, and the mean ΔCt value of less than ΔCt cut-off value for each sample was defined as TP53 K351N positive mutation. When the ΔCt measure exceeds ΔCt cut-off values for the mutant reactions, the assay reports no mutation detected.
Tumor cell lines Statistical analysis Human EOC A2780 cell line (ECACC catalog code: 93112519) and its cisplatin-resistant sub-line A2780CIS (ECACC catalog code: 93112517) were purchased from the Committee on Type Culture Collection of Chinese Academy of Sciences (CTCCCAS, Shanghai, China) and were routinely passaged in RPMI-1640 medium (GIBCO, Invitrogen). Patients and tumor samples For the present study, paraffin-embedded tumor samples of 153 patients with FIGO stage IIIC/IV EOC, consisting of 71 (46.41%) underwent
This study was performed in accordance with the criteria of reporting recommendations for tumour marker prognostic studies (REMARK) [28]. All statistical calculations were performed with the use of SPSS software (version 17.0). The positive rate of TP53 K351N mutation in patients with EOC was calculated by Clopper–Pearson with adjusted coefficient k (0.5). The relationship between TP53 K351N mutation status and DFS was analyzed with the Kaplan–Meier method with a log-rank (Mantel–Cox) test. Two-sided P value of less than 0.05 was considered to indicate statistical significance.
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Results
TP53 K351N mutation and survival analysis
Specificity and sensitivity of AS-PCR assay
The median DFS for NACT-IDS was 13.0 months (95% CI, 10.52 to 15.48 months) compared to 15.0 months (95%CI, 13.50 to 16.50 months) for PDS (Fig. 1A). The NACT-IDS patients exhibited significantly shorter DFS as compared with PDS patients (P = 0.02). When the 63 (88.7%, 63/71) patients who don't have TP 53 K351N mutation in the NACT-IDS group were compared to the 82 patients who underwent PDS, the median DFS for NACT-IDS and for PDS were 14 months (95% CI, 12.89 to 15.11 months) and 15 months (95% CI, 13.50 to 16.50 months), respectively (P = 0.10) (Fig. 1B). In multivariate analysis, TP53 K351N mutation is an independent factor for shorter DFS in the patients who underwent NACT-IDS (HR = 19.05; P = 0.01) (Fig. 1C and Table 1).
We tested the specificity and sensitivity of the allele-specific PCR assay for detecting TP53 K351N mutation in the positive control incorporated into the DNA template with wild-type allele. Our results showed the TP53 K351N missense mutation (AAG → AAT) of exon 10 in the positive control on DNA sequencing, and no overlap between the allele-specific primers and probes, and the signals of nonspecific hybridization and amplification in this AS-PCR assay (Supplementary Fig. 2A). A typical standard curve amplification plot and linear regression analysis (10−4 to 10−9 dilution of positive control) yielded a correlation coefficient of 0.992 and a y-intercept value of 24.49 and a slope of −3.783 which closely approximated the theoretical maximum amplification efficiency of 100% (−3.32 slope) (Supplementary Fig. 2A). The mean ΔCt value (4.84 ± 0.46, range 4.38–5.30) obtained by the subtraction of a CtHEX value of 10−9 from the CtFAM value of allelic reference was defined as the ΔCt cut-off value. For TP53 K351N, the mean ΔCt cut-off value of all genotypically mutant specimens for each assay ≤ 5 or N 5 was interpreted as mutation positive or mutation negative, respectively. Detection of TP53 K351N mutation in EOC cell lines This test is designed to detect platinum resistance-associated types of TP53 K351N mutation. To validate the results of this AS-PCR assay, we tested the suitability of this AS-PCR assay for detecting TP53 K351N mutation in cisplatin-resistant A2780CIS and cisplatin-sensitive A2780 cells previously identified as having the TP53 K351N mutation and as having wild-type alleles, respectively. This AS-PCR assay and sequencing identified the same TP53 K351N mutation in A2780CIS cells, whereas A2780 were confirmed as being negative, yielding a sensitivity of 96% and a specificity of 100%. This method could be used as a reliable test for TP53 K351N mutation. Identification of TP53 K351N mutations in tumors Using the AS-PCR assay, we analyzed paraffin-embedded tumors of 153 patients with stage IIIC/IV EOC previously treated with NACT-IDS and PDS followed by chemotherapy. In three independent runs, we identified TP53 K351N mutations (mean ΔCt N cut-off value) in 8 (11.27%, 95% CI, 4.98%–20.98%) of 71 NACT-IDS patients but not in 82 PDS patients. Platinum-based NACT exhibited a higher rate of TP53 K351N mutation, but the number of cycles did not significantly influence the rate of TP53 K351N mutation (P = 0.10). The accordant results between sequencing and AS-PCR assay were quantitatively and qualitatively verified (Supplementary Fig. 2B–D). TP53 K351N mutation and response to therapy Response to therapy was analyzed in the 153 patients who received platinum-based chemotherapy. The overall response rate to platinumbased chemotherapy in the NACT-IDS group was 80.28%, compared to an overall response rate of 82.92% in the PDS group (P = 0.42); 14 (19.72%) of 71 NACT-IDS patients relapsed within 6 months after completion of platinum-based chemotherapy, compared to 15 (18.29%) of 82 PDS patients (P = 0.49). However, in patients with relapse within 6 months after completion of NACT-IDS and PDS followed by chemotherapy, TP53 K351N mutation was observed in 8 of 14 NACT-IDS patients, and the rate of TP53 K351N mutation was significantly higher in NACT-IDS compared to PDS patients (57.14% vs. 0%, P b 0.01). In the 29 patients retreated with platinum-based chemotherapy at first recurrence within 6 months, 7 (50.00%), with TP 53 K351N mutation, of 14 patients initially treated with NACT exhibited a higher rate of progression while on therapy, compared to 2 (13.33%) of 15 patients treated with PDS (P = 0.04).
Discussion The development of chemoresistance prevents the efficient treatment of recurrent and progressive ovarian cancer. Despite advances in surgery and therapy, overall prognosis remains relatively poor, and relapse within 6 months and progression to chemotherapy resistance are frequently seen in many patients with advanced EOC [29]. It has been evidenced that EOC patients retreated with platinum-based chemotherapy at first recurrence after initial NACT-IDS exhibited a higher rate of progression and had platinum resistant disease at interval to second recurrence less than 6 months [13,30]. A better understanding of what it is about ovarian cancer that allows it to be platinum resistant to further therapy after an initial platinum-based NACT is needed for better treatment strategies that could circumvent platinum resistance. Both intrinsic and acquired resistance result from the numerous genetic and epigenetic changes occurring in cancer cells. Resistance to chemotherapy is believed to occur by two mechanisms: either selection of a population of tumor cells inherently resistant to the agents employed in treatment or the induction of resistance of these tumor cells by the chemotherapeutics themselves [31]. Some studies have postulated that a small subpopulation of intrinsic resistant cancer cells can continue to grow after treatment and will repopulate the tumor bulk, owing to the removal of competing or sensitive cells, whereas other studies have proposed that genetic changes or mutations may confer acquired cisplatin resistance during cancer chemotherapy [32]. However, whether resistance mutations have any selective advantage in the absence of therapy, or whether they are merely low-frequency events until the application of chemotherapy needs to be further investigated. Various lines of evidence indicate that cancer-associated TP53 mutations can contribute actively to tumor progression, increased resistance to platinum-based chemotherapy and shortened survival in ovarian cancer [18,19]. However, a review of the extensive literature on TP53 in ovarian cancer does not support a consistent relationship between TP53 mutation and response to therapy or survival in multivariate analysis [18,20]. This may reflect the complexity that may be an indication that mutations in the coding sequence represent only a subset of the functional TP53 alterations. The TP53 K351N mutation is believed to be linked to acquisition of platinum-resistance in ovarian cancer A2780 cells following cisplatin treatment. Mutations of the nuclear export signal (NES, Met340– Lys351) in the TD prevent TP53 nuclear export and tetramerization that are indispensable for TP53/BAX/caspases signaling-mediated cellular apoptosis [19,25]. The TP53 K351N mutant greatly reduced the ability to interact with BAK and BAK oligomerization, then leading to resistance to cisplatin-induced apoptosis [25,26]. It has been shown that in ovarian cancers, the resistance to cisplatin is due to the loss of mismatch repair (MMR) of DNA capable of increasing mismatch mutation rates at the specific genes, including TP53 gene, that control response to chemotherapy [33,34]. As cisplatin is itself a mutagen, it may aid the process of creating and developing a mutant
G. Zhang et al. / Gynecologic Oncology 132 (2014) 752–757 Fig. 1. Correlation between the TP53 K351N mutations in tumor specimen and shortened disease-free survival. (A) DFS for NACT-IDS vs. PDS. (B) DFS for NACT-IDS excluded the TP53 K351N mutant patients vs. PDS. (C) In NACT-IDS patients, the TP53 K351N mutation was associated with shortened DFS.
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Table 1 Cox regression analysis for recurrence within six months after NACT-IDS (N = 71). Disease-free survival
Univariate
Variables
HR(95%CI)
P value
Multivariate
Age (≥55 years vs b55 years) Histological grade (poor vs well/moderate) Malignant cells in ascites (yes vs no) Lymph node metastasis (yes vs no) Liver or lung metastasis (yes vs no) Residual tumor (N1 vs b1) NACT (N3 vs ≤3) K351N (yes vs no)
1.84 (0.62–5.50) 2.40 (0.75–7.65) 1.71 (0.38–7.67) 0.64 (0.18–2.28) 0.23 (0.08–0.70) 1.06 (0.37–3.06) 2.01 (0.67–5.99) 13.96 (4.82–40.47)
0.27 0.14 0.48 0.49 0.01 0.91 0.21 b0.01
HR(95%CI)
P value
1.47 (0.17–12.59)
0.73
19.05(2.37–152.77)
0.01
Abbreviations: DFS = disease-free survival; HR = hazard ratio; CI = confidence interval. Bold values indicate P b 0.05.
cell that is itself resistant to the treatment process and is a possible origin of platinum-resistant ovarian cancer cells following exposure to cisplatin [35–38]. Therefore, we wonder whether the platinum resistance disease in advanced EOC after platinum-based NACT-IDS arises owing to TP53 K351N mutations that develop during or following the first treatment or that pre-exist within the tumour bulk at the time of first treatment in a clinical setting. In this study, we showed for the first time that occurrence of TP53 K351N mutation after platinum-based NACT correlates with the platinum resistance of EOC in a clinical setting; We found that after initial treatment, when multivariate logistic regression was used to control for factors independently associated with recurrence within 6 months, NACT-IDS did not appear significantly to influence the recurrence rates within 6 months compared to PDS (P = 0.49). However, when response to treatment was evaluated in the 29 patients retreated with platinum-based chemotherapy at first recurrence within 6 months, half of the patients initially treated with NACT, having TP53 K351N mutation, exhibited an extremely poor response to the second-line platinum-based chemotherapy, with interval to second recurrence less than 6 months (P = 0.04), as reported in Ref.13. In fact, we also found that TP53 K351N mutations occurred in more than half of the NACTIDS patients relapsed within 6 months, suggesting that loss of MMR results in rapid evolution of platinum resistance during sequential rounds of drug exposure [31,34]. Moreover, when excluding the TP53 K351N mutation-positive patients, the difference in DFS between NACT-IDS and PDS was lost (P = 0.10) (Fig. 1B). Actually, in multivariate analysis, the TP53 K351N mutation as an independent factor significantly influenced DFS of EOC patients who received NACT (P b 0.001) (Fig. 1C and Table 1). Our data support the emergence of resistance mutations following treatment in some cases of relapsed ovarian carcinoma, since platinum-resistance disease at second relapse in those patients who received NACT-IDS occurs more frequently than does in those patients who received PDS, suggesting that NACT administration, when tumor burden is large and heterogeneous, may induce TP53 K351N mutant clones resulting in resistance of these tumor cells to platinum therapy through spontaneously mutant selection or cellular selfdefense mechanism resulting from genetic changes [39,40]. Our findings may explain at least to some extent that TP53 K351N mutation, which is distinct from mutations in other functional domains of the TP53 gene, plays a crucial role in conferring platinum resistance to second-line platinum-based chemotherapy after NACT in advanced EOC. The collection of sequential samples from patients before and after platinum-based chemotherapy will be critical for detection of small populations of TP53 K351N mutant subclones as even with nextgeneration sequencing. Based on TP53 K351N mutation, the selection of non-platinum-based chemotherapy for further treatment after platinum-based NACT-IDS would improve the outcome of NACT-IDS followed by chemotherapy. In conclusion, our study indicates that the TP53 K351N mutation may be associated with induction of platinum resistance after platinum-based
NACT in advanced EOC. As we would expect, a prospective clinical study is necessary to get reliable data on the TP53 K351N mutation-associated platinum resistance in ovarian cancer cells after platinum-based NACT. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.ygyno.2014.01.028.
Conflict of interest statement The authors have no conflicts of interest to declare.
References [1] Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA Cancer J Clin 2013;63(1): 11–30. [2] Vasey PA, Herrstedt J, Jelic S, Force EGT. ESMO Minimum Clinical Recommendations for diagnosis, treatment and follow-up of epithelial ovarian carcinoma. Ann Oncol 2005;16(Suppl. 1):i13–5. [3] Perez RP, Hamilton TC, Ozols RF, Young RC. Mechanisms and modulation of resistance to chemotherapy in ovarian cancer. Cancer 1993;71(4 Suppl.):1571–80. [4] Kelland L. The resurgence of platinum-based cancer chemotherapy. Nat Rev Cancer 2007;7(8):573–84. [5] Cornelis S, Van Calster B, Amant F, Leunen K, van der Zee AG, Vergote I. Role of neoadjuvant chemotherapy in the management of stage IIIC-IV ovarian cancer: survey results from the members of the European Society of Gynecological Oncology. Int J Gynecol Cancer 2012;22(3):407–16. [6] Worley Jr MJ, Guseh SH, Rauh-Hain JA, Williams KA, Muto MG, Feltmate CM, et al. Does neoadjuvant chemotherapy decrease the risk of hospital readmission following debulking surgery? Gynecol Oncol 2013;129(1):69–73. [7] Morrison J, Haldar K, Kehoe S, Lawrie TA. Chemotherapy versus surgery for initial treatment in advanced ovarian epithelial cancer. Cochrane Database Syst Rev 2012;8:CD005343. [8] Vergote I, Trope CG, Amant F, Kristensen GB, Ehlen T, Johnson N, et al. Neoadjuvant chemotherapy or primary surgery in stage IIIC or IV ovarian cancer. N Engl J Med 2010;363(10):943–53. [9] Vergote I, Trope CG, Amant F, Ehlen T, Reed NS, Casado A. Neoadjuvant chemotherapy is the better treatment option in some patients with stage IIIc to IV ovarian cancer. J Clin Oncol 2011;29(31):4076–8. [10] Vergote I, Amant F, Kristensen G, Ehlen T, Reed NS, Casado A. Primary surgery or neoadjuvant chemotherapy followed by interval debulking surgery in advanced ovarian cancer. Eur J Cancer 2011;47(Suppl. 3):S88–92. [11] Chi DS, Musa F, Dao F, Zivanovic O, Sonoda Y, Leitao MM, et al. An analysis of patients with bulky advanced stage ovarian, tubal, and peritoneal carcinoma treated with primary debulking surgery (PDS) during an identical time period as the randomized EORTC-NCIC trial of PDS vs neoadjuvant chemotherapy (NACT). Gynecol Oncol 2012;124(1):10–4. [12] Jelovac D, Armstrong DK. Recent progress in the diagnosis and treatment of ovarian cancer. CA Cancer J Clin 2011;61(3):183–203. [13] Rauh-Hain JA, Nitschmann CC, Worley Jr MJ, Bradford LS, Berkowitz RS, Schorge JO, et al. Platinum resistance after neoadjuvant chemotherapy compared to primary surgery in patients with advanced epithelial ovarian carcinoma. Gynecol Oncol 2013;129(1):63–8. [14] Agarwal R, Kaye SB. Ovarian cancer: strategies for overcoming resistance to chemotherapy. Nat Rev Cancer 2003;3(7):502–16. [15] Skilling JS, Squatrito RC, Connor JP, Niemann T, Buller RE. p53 gene mutation analysis and antisense-mediated growth inhibition of human ovarian carcinoma cell lines. Gynecol Oncol 1996;60(1):72–80. [16] Laframboise S, Chapman W, McLaughlin J, Andrulis IL. p53 mutations in epithelial ovarian cancers: possible role in predicting chemoresistance. Cancer J 2000;6(5): 302–8. [17] Gadducci A, Di Cristofano C, Zavaglia M, Giusti L, Menicagli M, Cosio S, et al. P53 gene status in patients with advanced serous epithelial ovarian cancer in relation to response to paclitaxel- plus platinum-based chemotherapy and long-term clinical outcome. Anticancer Res 2006;26(1B):687–93.
G. Zhang et al. / Gynecologic Oncology 132 (2014) 752–757 [18] Reles A, Wen WH, Schmider A, Gee C, Runnebaum IB, Kilian U, et al. Correlation of p53 mutations with resistance to platinum-based chemotherapy and shortened survival in ovarian cancer. Clin Cancer Res 2001;7(10):2984–97. [19] Perego P, Giarola M, Righetti SC, Supino R, Caserini C, Delia D, et al. Association between cisplatin resistance and mutation of p53 gene and reduced bax expression in ovarian carcinoma cell systems. Cancer Res 1996;56(3):556–62. [20] Ahmed AA, Etemadmoghadam D, Temple J, Lynch AG, Riad M, Sharma R, et al. Driver mutations in TP53 are ubiquitous in high grade serous carcinoma of the ovary. J Pathol 2010;221(1):49–56. [21] Muscolini M, Montagni E, Caristi S, Nomura T, Kamada R, Di Agostino S, et al. Characterization of a new cancer-associated mutant of p53 with a missense mutation (K351N) in the tetramerization domain. Cell Cycle 2009;8(20):3396–405. [22] Turanek J, Kasna A, Zaluska D, Neca J, Kvardova V, Knotigova P, et al. New platinum(IV) complex with adamantylamine ligand as a promising anti-cancer drug: comparison of in vitro cytotoxic potential towards A2780/cisR cisplatinresistant cell line within homologous series of platinum(IV) complexes. Anticancer Drugs 2004;15(5):537–43. [23] Behrens BC, Hamilton TC, Masuda H, Grotzinger KR, Whang-Peng J, Louie KG, et al. Characterization of a cis-diamminedichloroplatinum(II)-resistant human ovarian cancer cell line and its use in evaluation of platinum analogues. Cancer Res 1987;47(2):414–8. [24] Masuda H, Ozols RF, Lai GM, Fojo A, Rothenberg M, Hamilton TC. Increased DNA repair as a mechanism of acquired resistance to cis-diamminedichloroplatinum (II) in human ovarian cancer cell lines. Cancer Res 1988;48(20):5713–6. [25] Muscolini M, Montagni E, Palermo V, Di Agostino S, Gu W, Abdelmoula-Souissi S, et al. The cancer-associated K351N mutation affects the ubiquitination and the translocation to mitochondria of p53 protein. J Biol Chem 2011;286(46):39693–702. [26] Kamada R, Nomura T, Anderson CW, Sakaguchi K. Cancer-associated p53 tetramerization domain mutants: quantitative analysis reveals a low threshold for tumor suppressor inactivation. J Biol Chem 2011;286(1):252–8. [27] Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R, et al. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer 2009;45(2):228–47. [28] McShane LM, Altman DG, Sauerbrei W, Taube SE, Gion M, Clark GM. Statistics Subcommittee of the NCIEWGoCD: reporting recommendations for tumor marker prognostic studies. J Clin Oncol 2005;23(36):9067–72.
757
[29] Vergote I, Rustin GJ, Eisenhauer EA, Kristensen GB, Pujade-Lauraine E, Parmar MK, et al. Re: new guidelines to evaluate the response to treatment in solid tumors [ovarian cancer]. Gynecologic Cancer Intergroup. J Natl Cancer Inst 2000;92(18): 1534–5. [30] Bristow RE, Eisenhauer EL, Santillan A, Chi DS. Delaying the primary surgical effort for advanced ovarian cancer: a systematic review of neoadjuvant chemotherapy and interval cytoreduction. Gynecol Oncol 2007;104(2):480–90. [31] Cooke SL, Brenton JD. Evolution of platinum resistance in high-grade serous ovarian cancer. Lancet Oncol 2011;12(12):1169–74. [32] Galluzzi L, Senovilla L, Vitale I, Michels J, Martins I, Kepp O, et al. Molecular mechanisms of cisplatin resistance. Oncogene 2012;31(15):1869–83. [33] Branch P, Masson M, Aquilina G, Bignami M, Karran P. Spontaneous development of drug resistance: mismatch repair and p53 defects in resistance to cisplatin in human tumor cells. Oncogene 2000;19:3138–45. [34] Lin X, Howell SB. DNA mismatch repair and p53 function are major determinants of the rate of development of cisplatin resistance. Mol Cancer Ther 2006;5(5): 1239–47. [35] Xiao X, Melton DW, Gourley C. Mismatch repair deficiency in ovarian cancer— molecular characteristics and clinical implications. Gynecol Oncol 2014;132(2): 506–12. [36] Aebi S, Kurdi-Haidar B, Gordon R, Cenni B, Zheng H, Fink D, et al. Loss of DNA mismatch repair in acquired resistance to cisplatin. Cancer Res 1996;56(13): 3087–90. [37] de las Alas MM, Aebi S, Fink D, Howell SB, Los G. Loss of DNA mismatch repair: effects on the rate of mutation to drug resistance. J Natl Cancer Inst 1997;89(20): 1537–41. [38] Gercel-Taylor C, Scobee JJ, Taylor DD. Effect of chemotherapy on the mutation frequency of ovarian cancer cells at the HPRT locus. Anticancer Res 2005;25(3B): 2113–7. [39] Righetti SC, Perego P, Corna E, Pierotti MA, Zunino F. Emergence of p53 mutant cisplatin-resistant ovarian carcinoma cells following drug exposure: spontaneously mutant selection. Cell Growth Differ 1999;10(7):473–8. [40] Shen DW, Pouliot LM, Hall MD, Gottesman MM. Cisplatin resistance: a cellular selfdefense mechanism resulting from multiple epigenetic and genetic changes. Pharmacol Rev 2012;64(3):706–21.