Introduction
The plethora of comprehensive gene expression analyses in the context of breast cancer has gradually helped unravel the molecular biology of breast cancer. In addition, a large number of gene expression profiles that predict prognosis, recurrence, and therapeutic response to anticancer drugs and endocrine therapies has been reported [
1]. Representative gene expression profiles, such as Onocotype DX [
2‐
4], Mammaprint [
5,
6], and Prosigna [
7,
8], have already been approved by US Food and Drug Administration.
Tumor suppressor gene
TP53 is the most frequently mutated gene in human cancers, and the patients with
TP53 mutations are known to have poor clinical outcomes [
9]. Several large-scale meta-analyses have shown
TP53 mutation to be an independent predictor of poor prognosis for breast cancer [
10,
11]. Furthermore,
TP53 status is a predictive factor for chemotherapy [
12,
13].
We had earlier found a gene expression signature (
TP53 signature) that correlates with presence or absence of
TP53 mutation [
14]. The
TP53 status determined using the
TP53 signature was a prognostic factor independent of other known clinicopathological prognostic factors. Also, the
TP53 status determined using gene expression signature was a superior predictor of prognosis compared with that determined using immunohistochemical examination and direct DNA sequencing. Similar results were earlier reported by Miller et al. [
15].
The purpose of this study was to develop a simple diagnostic system for TP53 signature using multiplex reverse transcription–polymerase chain reaction (RT–PCR), to test its diagnostic precision and prognostic predictability in a prospective cohort and to examine the clinical significance of TP53 signature among breast cancer subtypes.
Patients and methods
Patients and tumor tissues
This study was approved by the Ethics Committee at the Tohoku University Hospital (TU), Hoshi General Hospital (HG), and Miyagi Cancer Center (MCC). The TU cohort, which was used in our previous study [
14], was used for the development of the
TP53 signature diagnosis system. Validation cohort is a breast cancer case series from HG and MCC prospectively from September, 2007 to October, 2013 [
16]. None of the cases received chemotherapy or endocrine therapy preoperatively. Written informed consent for the study was obtained from all patients. A part of the surgical specimen of breast cancer was stored as fresh frozen (FF) tissue and/or formalin-fixed paraffin embedded (FFPE) tissue. Among patients enrolled in this study, we selected curatively resected patients with stage I–II breast cancer. Patients with ductal carcinoma in situ, those with unknown histology or those with squamous cell carcinoma were excluded from the analysis. The validation cohort was used to assess the prognostic ability of the
TP53 signature diagnosis system.
Clinicopathological characteristics
Clinicopathological characteristics data (pathological tumor size, pathological lymph node status, pathological stage, ER, PgR, HER2, Grade, Ki-67, adjuvant chemotherapy and adjuvant endocrine therapy) were obtained from medical records. For cases for which Ki-67 data were not available, immunohistological staining for Ki-67 was performed at the Department of Pathology, Tohoku University Hospital, using the MIB-1 antibody (Dako, Carpinteria, CA, USA).
The glass slide specimen with 10-µm thick sections of FF and FFPE tissue blocks were prepared. In reference to the HE stained specimen, tumor cells were collected from FF tissue or FFPE tissue by macrodissection technique. Total RNA was extracted from FF tissue or FFPE tissue with use of RNeasy mini kit (Qiagen, Valencia, CA, USA) or RNeasy FFPE kit (Qiagen, Valencia, CA, USA), respectively.
TP53 signature diagnosis system
Genome Lab GeXP Genetic Analysis System (Beckman Coulter, Brea, CA) was used to obtain gene expression profile. To obtain the
TP53 signature gene set for GeXP, genes for which the average signal value in the raw data exceeded 1000 in the previous microarray data of the TU cohort [
14] and which had less homolog genes were selected. Based on these criteria, 23 genes were chosen among
TP53 signature genes. Three genes were added to this gene set as internal control; as a result, a
TP53 signature gene set that comprised of 26 genes was established (Supplemental Table 1). Primers for reverse transcription (RT) and for PCR were designed using Genome Lab eXpress Designer GeXP Software (Beckman Coulter, Brea, CA). The multiplex reaction was optimized as per the manual and optimal primer concentrations determined. RT and PCR were performed with GenomeLab GeXP Start Kit (Beckman Coulter, Brea, CA) in accordance to the manual. The quantity of input RNA was 1 μg for FFPE tissues and 50 ng for the FF tissues.
TP53 signature score
TP53 status was determined by
TP53 signature score, which is the ratio of the sum of expression levels of 16 genes that were upregulated in tumors with
TP53 mutation to the sum of expression values of 7 genes downregulated in tumors with
TP53 mutation. The cutoff level for
TP53 signature score was determined by Receiver Operating Characteristic curve (ROC) analysis based on the
TP53 signature status by microarray of TU cohort [
14]. When
TP53 signature score of a certain sample was greater than 1.11, the sample was labeled as
TP53 mutant signature.
Outcomes
The primary end point of the study was recurrence-free survival (RFS), which was defined as the period from the date of surgery for breast cancer to the date on which tumor recurrence. Overall survival (OS) was defined as the period from the date of surgery for breast cancer to the date of death. Breast cancer-specific survival (BCSS) was defined as the period from the date of surgery for breast cancer to the date of death by breast cancer.
Statistical analysis
All statistical analyses were performed using JMP Pro 14.3.0 (SAS Institute Japan Co., Ltd., Tokyo, Japan). Baseline characteristics of patients (except age) were assessed by chi-squared test. Kruskal–Wallis test was used for statistical analysis of age. Survival curves were made with Kaplan–Mayer method, and between-group differences assessed with log-rank test. Univariate and multivariate analyses (Cox proportional hazard model) were conducted to assess the association between clinicopathological factors and the
TP53 status for RFS. P value under 0.05 was considered indicative of a statistically significant difference. This study is registered in UMIN-CTR (
http://www.umin.ac.jp/ctr/) (000005172).
Discussion
The
TP53 mutation has long been known as an independent predictor of poor prognosis among patients with breast cancer [
10,
11]. To develop a reliable diagnostic kit, we created the gene expression signature that could diagnose the
TP53 gene status using microarray analysis [
14]. Uji et al. reported that the
TP53 status determined by gene expression signature was a superior predictor of prognosis than
TP53 status determined on direct DNA sequencing (including the classical Sanger sequencing and the NGS method) [
17]. Today, although the
TP53 gene mutation can be analyzed in detail by the cancer genome profiling test, the
TP53 signature is considered to have an advantage in terms of prognosis prediction for breast cancer. Lehmann et al. verified the prognostic predictability of 351 reported gene expression profiles on a meta-analysis based on 31 breast cancer cohorts [
18]. They found
TP53 signature was a robust prognostic factor, and was better than well-known gene expression profiles such as OnctypeDX and Mammaprint. Furthermore, Lehmann et al. verified that
TP53 signature was a predictor of therapeutic response in their meta-analysis [
18]. Similarly, Oshima et al. reported that signature could predict response to preoperative chemotherapy [
19]. As described above,
TP53 signature is confirmed to be both an independent prognostic factor and an independent predictor for response to chemotherapy.
In this report, a simple and easy multiplex RT–PCR diagnostic system for
TP53 signature was developed and the rate of agreement of
TP53 status by
TP53 signature score and the
TP53 status by microarray was enough high (97.1%) (Table
1).
In the validation cohort, a significant difference was observed between the two
TP53 signatures with respect to ER, PgR, HER2, histological grade, Ki-67 histological type, adjuvant chemotherapy and adjuvant endocrine therapy (Table
2). These results do not contradict those reported from previous studies [
14,
15,
20,
21].
The TP53 mutant signature based on the TP53 signature score was associated with significantly poor RFS, OS and BCSS as compared to that associated with the TP53 wild-type signature. On univariate and multivariate analysis, TP53 signature was significantly associated with PFS independent of other clinicopathological factors. These results indicate that the TP53 status diagnosed by this diagnostic system was an independent prognostic factor in patients with breast cancer for whom curative resection (stage I–II) is performed.
In this report, we showed for the first time that there was clinical significance among breast cancer subtypes and grades. In the ER positive, especially in Luminal B like subgroup, Grade1 and 3 subgroup, it was clearly seen that the prognosis was closely associated with the TP53 status. In ER negative group, Luminal A like subtype and TNBC, the significant difference was not observed between TP53 signature status. But, because there was no recurrence in TP53 wild-type signature group, it can be said that TP53 signature had clinical significance in these subtypes.
There are some limitations of the interpretation of this study. First, the sample size was relatively small, and the recurrence events were few so far. We are going to follow up recurrent events sequentially. Second, uniform treatment intervention was not carried out for the study cohort because it is an observational, prospective study. We are currently conducting a large scare retrospective-prospective study to confirm the clinical significance of TP53 signature using several prospective studies conducted in Japan.
In conclusion, we developed a relatively simple multiplex RT–PCR diagnostic system to determine the TP53 signature. Its diagnostic accuracy and prognostic value were verified in a prospective cohort. And we showed the clinical significance of TP53 signature among breast cancer subtypes. This simple and precise diagnostic system may help in prognostic assessment, therapeutic decision-making, and treatment optimization in patients with breast cancer.
Declarations
Conflict of interest
Dr. Takahashi reports personal fees from Taiho, Chugai, Asahikasei, Bayer, Japan blood products organization, Medicon, Termo, Sanofi, Nippon-kayaku, Takeda, Yakult, grants and personal fees from Merckbiopharma, grants from Ono, outside the submitted work; In addition, Dr. Takahashi has a patent JP4370409B2 issued. Dr. Ishida reports grants from Taiho, Eisai and Kyowa-Kirin, grants and personal fees from Chugai, personal fees from Pfizer and Astra Zeneca, outside the submitted work. Prof. Ishioka reports grants and personal fees from Novartis, Daiichi Sankyo, Bayer, Bristol-Myers Squibb, Nippon-Kayaku, Hitachi, personal fees from Taiho, Ono, Merckbiopharma, AsahiKasei, Sanofi, Takeda, Eisai, Eli Lilly, Mundipharma, Teijin, Chugai, Konica Minolta, Pfizer, Mochida, grants from Riken Genesis, MSD, Linical, outside the submitted work; In addition, Prof. Ishioka has a patent JP4370409B2 issued. All the remaining authors have no conflicts of interest.
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