Detection of TP53 and PIK3CA Mutations in Circulating Tumor DNA Using Next-Generation Sequencing in the Screening Process for Early Breast Cancer Diagnosis

Circulating tumor DNA (ctDNA) has emerged as a non-invasive “liquid biopsy” for early breast cancer diagnosis. We evaluated the suitability of ctDNA analysis in the diagnosis of early breast cancer after mammography findings, comparing PIK3CA and TP53 mutations between tumor biopsies and pre-biopsy circulating DNA. Matched plasma and frozen fresh tissue biopsies from patients with Breast Imaging-Reporting and Data System (BIRADS) 4c/5 mammography findings and subsequent diagnosis of primary breast cancer were analyzed using NGS TruSeq Custom Amplicon Low Input Panel (Illumina) and plasma SafeSEQ (Sysmex Inostics). The same plasma and tumor mutations were observed in eight of 29 patients (27.6%) with four in TP53 and five in PIK3CA mutations. Sequencing analysis also revealed four additional ctDNA mutations (three in TP53 and one in PIK3CA) previously not identified in three patients tissue biopsy. One of these patients had mutations in both genes. Age, tumor grade and size, immunohistochemical (IHC) subtype, BIRADS category, and lymph node positivity were significantly associated with the detectability of these blood tumor-derived mutations. In conclusion, ctDNA analysis could be used in early breast cancer diagnosis, providing critical clinical information to improve patient diagnosis.


Introduction
Breast cancer (BC) is the most common cancer in females worldwide, the second most common cause of death from cancer among males and females (only preceded by lung cancer), and a leading cause of premature mortality from cancer as measured by average and total years of life lost [1].

Study Design and Patient Population
The pilot study included 29 patients with BIRADS 4c/5 mammography findings and subsequent diagnosis of primary breast cancer recruited at Virgen de la Victoria Hospital Málaga and Clinic University Hospital in Valencia. Blood samples were collected immediately (less than an hour) before tissue biopsy and prior to receiving any type of treatment. The matched tumor tissues were collected through core needle biopsies of breast tumors, which were subsequently fresh-frozen. Immunohistochemical (IHC) analysis was performed to quantify expression of human epidermal growth factor receptor 2 (HER2), hormone receptors (HR) and Ki67. Estrogen receptor (ER) and progesterone receptor (PR) were considered positive if tumors had more than 1% nuclear-stained cells. HER2 staining was scored on a scale of 0 to 3+, according to the HercepTest guidelines [24]. HER2 status was considered positive when graded as 3+, while 0 to 1+ were negative and 2+ was an inconclusive result and silver in situ hybridization (SISH) was performed in those cases to confirm positivity. Hormone receptor positive tumor plus Ki 67 index of <14% was considered a luminal A tumor while >14% was considered luminal B. IHC breast cancer subtypes were defined using a combination of these IHC markers as follows: Luminal A (ER-positive and/or PR-positive, HER2-negative and Ki-67 < 14%), luminal B (ER-positive and/or PR-positive, HER2-negative and Ki-67 ≥ 14%), HER2 positive (ER negative, PR negative, HER2 positive), and triple negative (ER, PR, and HER2 negative).
The study was approved by the research ethics committees at Virgen de la Victoria Hospital and Clínico de Valencia Hospital, all patients provided written informed consent. Research was conducted according to Good Clinical Practice and the Declaration of Helsinki.

DNA Extraction
Tumor DNA was isolated from fresh frozen tissue samples using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA) following the manufacturer's instructions. Plasma blood samples of 10 mL were collected in STRECK tubes immediately before the tissue biopsy. Within 2 h after collection, plasma was separated from whole blood samples through centrifugation for 10 min at 3000 rpm at room temperature and stored at −80 • C until further use. The plasma samples were defrosted and were centrifuged again for 10 min at 13,000 rpm at room temperature prior to DNA extraction to remove debris. Isolation of DNA from 2 mL of plasma was performed using QIAamp Circulating Nucleic Acid Kit (Qiagen) according to the manufacturer's instructions and eluted in 140 µL of AVE buffer (Qiagen). The total amount of amplifiable human genomic DNA isolated from plasma samples was quantified using a modified version of human long interspersed element 1 (LINE-1) real-time PCR assay and reported as genome equivalents (GE) [25,26].

Targeted Sequencing
The NGS study on fresh frozen tissue samples was performed using a customized design of TruSeq Custom Amplicon Low Input Panel (Illumina), which includes the full region of the PIK3CA and TP53 genes (the most commonly mutated genes in early-stage BC according to TCGA). Libraries were constructed using Tru-Seq reagents from Illumina according to the standard protocol provided by Illumina. All DNA samples were diluted to the same initial concentration (25 ng/µL). In order to artificially increase the genetic diversity, 10% DNA from phage PhiX was added to the library of genomic DNA before loading on the flow-cell. Exon regions were captured in a solution using the Agilent SureSelect v.5 kit according to the manufacturer's instructions (Agilent, Santa Clara, CA, USA). Paired-end sequencing was performed using a HiSeq 2500 Genome Analyzer (Illumina, San Diego, CA, USA). The sequences were aligned to the human genome reference sequence (hg19) using the Eland algorithm of CASAVA 1.8 software (Illumina, San Diego, CA, USA). The chastity filter of the BaseCall software of Illumina was used to select sequence reads for subsequent analysis. The ELANDv2 algorithm of CASAVA 1.8 software (Illumina, San Diego, CA, USA) was then applied to identify point mutations and small insertions and deletions. Potential somatic mutations were filtered and manually curated.

Plasma DNA Sequencing (SafeSEQ)
Plasma sequencing was performed using Plasma SafeSEQ (Sysmex Inostics, Hamburg, Germany). SafeSEQ analysis was performed on up to 10,000 GE (~33 ng DNA) per sample. Briefly, human genomic DNA isolated from plasma samples was amplified (15 cycles) in 10 replicate PCR wells using primers containing unique identifier sequences (UIDs), which consisted of 14 random bases with an equal probability of A, C, T, and G, to allow for the distinction of each template molecule. The amplified reactions were purified using AMPure XP beads (Beckman Coulter) and eluted in 250 mL of Buffer EB (Qiagen). A fraction of purified PCR product was then amplified in the second round of PCR with universal primers. The PCR products were purified with AMPure XP and sequenced on an Illumina MiSeq instrument. High-quality sequence reads were selected based on quality scores, which were generated by the Illumina sequencing instrument to indicate the probability that an error was made in base calling. The template-specific portion of the reads was matched to reference sequences. Reads from a common template molecule were then grouped based on the UIDs that were incorporated as molecular barcodes. Variants calls from the SafeSEQ assay were considered real if ≥90% of the PCR fragments with the same UID contain an identical mutation. Wells with fewer than 200 UIDs as a result of poor amplification were excluded from analysis. Once the analysis was performed, the following criteria were followed to call a mutation: (1) Variant allele frequency (VAF) of at least 0.05% and (2). The mutation needed to be detected in at least two out of 10 replicate wells.

Statistical Analysis
The statistical analysis was performed with SPSS (19.0, SPSS Inc., Chicago, IL, USA). For association analyses between clinicopathological variables and PIK3CA and TP53 derived-mutations in blood, Spearmen correlation and Fisher's exact test (for categorical variables) were used. The threshold for statistical significance was set at p < 0.05.
Concerning the tumor grade, the majority of the patients presented grade 2 tumors (16 patients, 55%) while seven patients (24.5%) had grade 3 tumors and five (17%) patients had grade 1 tumors. The tumor grade was unknown in one patient (3.5%).

Analysis of PIK3CA and TP53 Mutations in Fresh Frozen Tissue Samples of Primary Breast
Mutations of the PIK3CA and TP53 genes were analyzed in genomic DNA from fresh frozen tissue samples of 29 primary breast cancer using the Illumina platform and the TruSeq Custom Amplicon Low Input Panel. Samples were successfully sequenced, and data quality was high, probably because DNA was isolated from fresh tissue material and had not been degraded. A total of 34 somatic mutations were detected in the 29 primary breast cancer patients. Of the 29 patients, 19 (65.5%) had PIK3CA mutations, six (20.7%) had TP53 mutations, and four (13.8%) had both PIK3CA and TP53 mutations. One (3.4%) patient had two PIK3CA mutations. In summary, all tumor samples were found to harbor at least one mutation.
In    One patient with a grade 3 luminal B tumor had mutations on both genes, TP53 (c.743G > T) and PIK3CA (c.3140A > T). One patient with a grade 2 luminal A tumor had two different PIK3CA mutations (c.1651C > A and c.1633G > A) and one patient with a grade 2 luminal A tumor had two different TP53 mutations (c.641A > G and c.748C > T) ( Table 3). The rest of TP53 mutations were mostly detected in patients with luminal B tumors (three of six (50%)), followed by one (16.7%) patient with triple-negative tumor and two (33.3%) patients with luminal A tumors. With respect to PIK3CA mutations, were detected mostly in patients with luminal A tumors (three of five (60%)), while two (40%) patients with PIK3CA mutations had luminal B tumors.   (Table 4).
Moreover, base transition (purine-pyrimidine and pyrimidine-purine) was more frequent than transversions (pyrimidine-pyrimidine and purine-purine). All these concordant somatic mutations were pathogenic, eight of them were missense mutations and one was a nonsense mutation.

Clinicopathological Variables Associated with the Detectability of Tumor-Derived PIK3CA and TP53 Mutations in Blood
We analyzed the association between the detectability of tumor PIK3CA and TP53 mutations in ctDNA and patient clinicopathological variables ( Table 5). The analysis indicated that the presence of ctDNA mutations was significantly associated with a lower age of the patients (p = 0.040), higher tumor size (p = 0.033) and tumor grade (p = 0.041), the presence of lymph node positivity (p < 0.001), a BIRADS category 5 (p = 0.004) and the IHC cancer subtype (p = 0.025).

Discussion
In this pilot study, we have demonstrated the possible implementation of plasma tumor DNA detection as noninvasive means for early breast cancer screening.
Data are currently limited about whether ctDNA analyses would be applicable to the screening process in early breast cancer detection, in part because the low tumor burden of early stage disease makes the detection of ctDNA difficult [18,20] and very low levels of plasma ctDNA are not normally detectable [25,27]. In our study and with the aim of improving this detection problem, the novel approach of SafeSEQ was employed. This technique is able to detect rare mutations with an allele frequency <0.001% [19].
In our population of 29 breast cancer screening patients, the NGS analysis of the tumor biopsies detected PIK3CA mutations in 79.3% (23/29) and TP53 in 34.5% (10/29) of primary breast cancer patients. While, one third (10/29, 34%) of the patients were found to have mutations in plasma samples. In total, 13 mutations were found, six (46.1%) different PIK3CA mutations and seven (53.8%) different TP53 mutations. The VAF in ctDNA was low (0.05%-3.60%) with the exception of one case with 20.56% in a patient later found to have lung metastasis in the CT scan performed as part of the staging process.
Of the 13 plasma mutations, nine were concordant with the mutations found in matched tissue samples (five PIK3CA and four TP53 mutations) in eight of 29 patients. Of these nine mutations, four PIK3CA mutations within the PI3Ka (p.E545K and p.E542K) and PI3Kc (p.H1047R and p.H1047L) have been previously reported as important hotspots in breast cancer [28][29][30][31]. These findings may highlight the potential role of ctDNA to capture the molecular abnormalities of the tumor at the very first stage of the disease, even before having a diagnosis.
On the other hand, in this study four additional mutation, three TP53 (c.641A > G, c.659A > G, c.748C > T) and one PIK3CA (c.1651C > A) not found in the tissue were found in plasma of three different patients, which did not have any undetected metastases or additional undiagnosed tumors. All these TP53 mutations have been reported in the catalog of somatic mutations in cancer (COSMIC) as driver mutations. This finding may be related to the capability of ctDNA to capture the tumor heterogeneity, which is indeed a limitation of tumor biopsies [32].
To date, a few applications of the liquid biopsies have been integrated into daily clinical practice, such as for molecular profiling of the tumor and monitoring resistance [33]. In the case of breast cancer, some studies have demonstrated its potential role in tracking therapeutic response and detection of relapse [14,34,35]. In a study with patients with more advanced locoregional disease, Riva et al. detected TP53 plasma mutations in 27 of 36 triple-negative patients (75%) before neoadjuvant chemotherapy treatment using dPCR [13]. Likewise, in another study PIK3CA (p.H1047R, p.E545K, and p.E542K) mutations in ctDNA were found in 22% of 110 stage I-III BC patients [36].
Two recent studies have also investigated the potential of liquid biopsy for the early detection of different type of cancer. Cohen et al. applied a screening method combining mutation detection in ctDNA with circulating protein markers in 1005 patients with nonmetastatic, clinically detectable tumors across eight common tumor types (ovary, liver, stomach, pancreas, esophagus, colorectal, lung, and breast). The median sensitivity of this test among the eight cancer types evaluated was 70% and ranged from 98% in ovarian cancers to 33% in breast cancers. At this sensitivity, the specificity was >99%, only seven of the 812 individuals without known cancers scored positive [37]. Chan et al. used detection of Epstein-Barr Virus (EBV) DNA in plasma to screen for nasopharyngeal carcinoma in 20.174 Chinese patients. Overall, the sensitivity and specificity of this approach were 97.1% and 98.6%, respectively [38].
In addition, several clinicopathological variables have been found associated with PIK3CA and TP53 plasma ctDNA mutation detectability in early breast cancer patients. We found a trend for a higher plasma ctDNA mutation burden in patients with clinical characteristics associated with more aggressive disease factors such as higher tumor grade (grade II-III), and tumor size, BIRADS category 5, the presence of positive lymph nodes and the IHC subtype present, being more frequently mutated in luminal A tumors followed by luminal B tumors. Similar results have been described in a previous study in primary breast cancer patients where clinicopathological features, such as N stage and hormone receptor status, were associated with the detectability of tumor-derived mutations in blood [39]. Then, this finding may help to explain the absence of ctDNA mutations for some patients in the early breast cancer screening.
Regarding clonal hematopoiesis, detectable clonal expansions most frequently involved somatic mutations in DNMT3A, ASXL1, and TET2 genes [40]. Furthermore, recent studies demonstrated that the genes more commonly associated with clonal hematopoiesis of indeterminate potential (CHIP) were DNMT3A, ASXL1, TET2, JAK2, SF3B1, CBL, GNAS, and IDH2 [41]. Interestingly, when healthy individuals were assessed for CHIP, no alterations in driver genes related to solid cancers were detected [42]. For these reasons, clonal hematopoiesis was not analyzed in our population, since TP53 and PIK3CA are commonly mutated genes in breast cancer.

Conclusions
In conclusion, to our knowledge, this is the first pilot study done in patients at the breast cancer screening process before undergoing any invasive diagnostic procedure or treatment, which shows that ctDNA analysis could be used in early breast cancer diagnosis. This study has demonstrated that ctDNA may reflect the PIK3CA and TP53 tumor-derived mutations present in very early breast cancer patients. Nevertheless, several clinicopathological variables can affect the detectability of these ctDNA mutations. Although our study population is small and larger-scale studies will be necessary to validate our results, we can conclude that early ctDNA testing can provide critical clinical information that may improve patient diagnosis. for his help with the English language version of the text.

Conflicts of Interest:
The authors declare no conflict of interest.