Comparison of the Mutation Proles of Triple-Negative Breast Cancers and Hormone Receptor-Positive/Human Epidermal Growth Factor Receptor 2-Negative Breast Cancers at the T2N0-1M0 Stage

Triple-negative breast cancer (TNBC) has higher loco-regional recurrence and visceral metastasis compared to other breast cancer subtypes; however, little is known about the molecular pathogenesis and therapeutic targets of TNBC. Therefore, we compared the mutation proles of early TNBC with those of hormone receptor-positive (HR+)/human epidermal growth factor receptor 2-negative (HER2 (cid:0) ) breast cancer using a customized next-generation sequencing capture panel. Methods DNA was obtained from the primary tumor tissues of 34 patients diagnosed with pT2N0-1M0 HR+/HER2 (cid:0) breast cancer or TNBC. To enrich the 48 breast cancer-associated genes, 21,192 probes were designed using the SureSelect design tool. After library preparation using the SureSelect XT kit (Agilent), paired-end DNA sequencing was performed on a HiSeq platform (Illumina). The mean depth of the target regions was 1,766 (×). The subsequent output containing genetic variation was analyzed using a pipeline of bioinformatics tools. Signicant mutations with allele frequencies of more than 30% were checked for their germline counterparts in the peripheral blood. Circulating cell-free nucleic acids were extracted and analyzed with a therascreen ® PIK3CA RGQ PCR kit (QIAGEN).


Abstract Background
Triple-negative breast cancer (TNBC) has higher loco-regional recurrence and visceral metastasis compared to other breast cancer subtypes; however, little is known about the molecular pathogenesis and therapeutic targets of TNBC. Therefore, we compared the mutation pro les of early TNBC with those of hormone receptor-positive (HR+)/human epidermal growth factor receptor 2-negative (HER2 ) breast cancer using a customized next-generation sequencing capture panel.
Methods DNA was obtained from the primary tumor tissues of 34 patients diagnosed with pT2N0-1M0 HR+/HER2 breast cancer or TNBC. To enrich the 48 breast cancer-associated genes, 21,192 probes were designed using the SureSelect design tool. After library preparation using the SureSelect XT kit (Agilent), paired-end DNA sequencing was performed on a HiSeq platform (Illumina). The mean depth of the target regions was 1,766 (×). The subsequent output containing genetic variation was analyzed using a pipeline of bioinformatics tools. Signi cant mutations with allele frequencies of more than 30% were checked for their germline counterparts in the peripheral blood. Circulating cell-free nucleic acids were extracted and analyzed with a therascreen ® PIK3CA RGQ PCR kit (QIAGEN).

Conclusions
The TP53 mutation is associated with higher tumor grade and Ki-67 expression in both groups, and with larger tumor size in TNBC, but not in HR+/HER2-breast cancer. In the foundation of TP53 mutation, concomitant mutation numbers are proportional to tumor size, re ecting clonal progression. Breast cancer-associated mutations such as those in TP53 and PIK3CA have different biological implications for the proliferation and clonal diversi cation of these two distinct groups of breast cancer.

Background
Triple-negative breast cancer (TNBC) has greater loco-regional recurrence and more distant metastasis compared to the other breast cancer subtypes [1][2][3]. Furthermore, TNBC lacks a targeted therapy because of the absence of the hormone receptor (HR) and human epidermal growth factor receptor 2 (HER2). Therefore, many researchers have tried to classify breast cancers according to molecular genetic events and sought to identify novel drug combinations by analyzing the accompanying gene mutations [4][5][6][7].
Approximately 40% of HR-positive breast cancers harbor PIK3CA mutations. Recently, the PI3K inhibitor alpelisib with fulvestrant received Food and Drug Administration (FDA) approval for PIK3CA-mutated HRpositive (+)/HER2-negative (−) advanced breast cancer after prior endocrine therapy [9]. The FDA also approved companion diagnostic tests for PIK3CA mutations for specimens of either tumor tissue or plasma cell-free nucleic acids [10].
Therefore, we analyzed breast cancer-associated gene mutations in tissues of diagnosed TNBC and HR+/HER2− breast cancer using a customized NGS panel and ran the therascreen® PIK3CA RGQ PCR assay (QIAGEN) with cell-free nucleic acids extracted from PIK3CA-mutated patients. Finally, the clinicopathological parameters and accompanying gene mutations of relatively early stage TNBC and HR+/HER2− breast cancer were compared.

Patients
This study included 34 Korean patients who were diagnosed with pT2N0-1M0 HR+/HER2− or triplenegative primary invasive ductal cancer at Pusan National University Yangsan Hospital, Korea, in 2016. They were treated with surgery and adjuvant chemotherapy, radiation therapy, or endocrine therapy. We assessed the histology, tumor grade, tumor size, lymph node status, type of surgery, and status of chemotherapy, radiation therapy, and endocrine therapy. We used Allred scoring to grade the hormone receptor status, with scores of 3 to 8 considered positive. HER2 was analyzed immunohistochemically, and the results were scored on a scale of 0, 1+, 2+, and 3+. When the result was 2 + and considered equivocal, silver in situ hybridization was performed. TNBC included tumors that were negative for hormone receptors and HER2. This study was approved by the Institutional Review Board of Pusan National University Yangsan Hospital.
Tumor DNA extraction and customized targeted sequencing Circulating cell-free nucleic acid extraction from plasma and PIK3CA testing using the therascreen® PIK3CA RGQ PCR assay Circulating cell-free nucleic acids were extracted with a QIAamp DSP Circulating Nucleic Acids kit (QIAGEN, Hilden, Germany) after double centrifugation from the plasma of 24 breast cancer patients including seven with PIK3CA mutations discovered by targeted sequencing. The quality of the extracted cell-free nucleic acids was checked using the 4150 TapeStation System (Agilent Technologies Inc.). The therascreen® PIK3CA RGQ PCR assay using real-time PCR (QIAGEN) was performed to detect C420R, E542K, E545A/D/G/K, Q546E/R, and H1047L/R/Y according to the manufacturer's instructions.
Sanger sequencing for determining germline/somatic mutations All TP53 and other pathogenic and likely pathogenic mutations with variant allele frequencies above 30% were checked for the presence of their germline counterparts by Sanger sequencing of available peripheral blood. We directly sequenced the genomic DNA using a BigDye™ Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA) and an ABI 3700 Genetic Analyzer (Applied Biosystems).

Statistical analysis
The tumor characteristics and gene mutations were compared using chi-square tests and Pearson correlations, and a p-value less than 0.05 was considered to denote signi cance. The data were analyzed using MedCalc (ver. 12.7.0.0, MedCalc Software, Ostend, Belgium). Table 1 shows the clinical and pathological characteristics of the enrolled breast cancer patients. All patients were diagnosed with invasive ductal cancer after surgery in 2016. Fourteen patients were diagnosed with pT2N0 and 20 with pT2N1. The median patient age was 50 (range 38-67) and 58 (range 37-75) years in the TNBC and HR+/HER2− groups, respectively. Although patient age did not differ signi cantly between groups, the age range in the TNBC group indicates more radiotherapy followed by breast-conserving surgery. Regarding the tumor grade, the majority of the TNBC patients had grade 3 tumors (11 patients, 91.7%), whereas 10 (45.5%) HR+/HER2− breast cancer patients had grade 3 tumors (p = 0.029). TNBC was closely related to p53 positivity and a high Ki-67 index (Table 1, Fig. 1). Conversely, lymphovascular invasion was more frequent in HR+/HER2− breast cancer. Loco-regional recurrence occurred in two patients one in each group. One patient with TNBC had lung and bone metastases during follow-up. Regarding primary tumor tissue mutations, nine patients had PIK3CA mutations and 14 had TP53 mutations. As expected from the immunohistochemistry, TNBC was associated with TP53 mutations detected by targeted sequencing (Table 1). germline mutations, co-existed with somatic PIK3CA, TP53, and ERBB2 mutations, and were found only in the HR+/HER2− group. However, only two of the four BRCA1 mutations were germline mutations; one was a frame-shift mutation (c.3627dupA) that was also changed to a homozygous mutation in tumor tissue by somatic loss of heterozygosity (LOH).

Patient characteristics
We analyzed the relationship between the TP53 and PIK3CA mutations based on tumor DNA and the patients' pathological parameters ( Table 2). The presence of TP53 mutations was associated with a higher tumor grade (p = 0.008), p53 positivity (p < 0.001), and a higher Ki-67 index (p = 0.004). The presence of PIK3CA mutations was associated with lymphovascular invasion (p = 0.018) and a lower Ki-67 index (p = 0.022). Comparison of tumor size according to TP53 and accompanying mutations Although every subject was classi ed as the same T2 stage (widest primary tumor size 2-5 cm), TP53mutated tumors were smaller than TP53-non-mutated ones in the HR+/HER2− group; however, TP53mutated tumors were relatively large in the TNBC group (Fig. 3A). In other words, the TP53-mutated TNBC tumors were larger than the TP53-mutated HR+/HER2− breast cancers (p = 0.0493) (Fig. 3A). Tumor size was correlated with the total number of signi cant mutations including TP53 and other accompanying mutations in TP53-mutated breast cancers (r = 0.7156, p = 0.004) (Fig. 3B).

Discussion
TNBC is clinically aggressive with a shorter interval to recurrence and metastasis compared to HR + breast cancer [1][2][3]11]. TNBC has no speci c targeted therapy other than surgery, chemotherapy, and radiotherapy because it does not express HR or HER2. However, high-throughput molecular diagnostic technologies such as NGS and microarray analyses have expanded our knowledge of the genetic alterations in cancers and provided new insight into the molecular pathogenesis of cancers [8,12,13]. Using NGS, the most commonly mutated genes in primary breast cancer are TP53 and PIK3CA [8]. The frequency of mutations in these genes depends on the breast cancer subtype. TNBC has a higher predominance of TP53 mutations compared to luminal A breast cancer [5,8,[14][15][16]. Similarly, we also found a higher incidence of TP53 mutations in TNBC (75.0%) compared to HR+/HER2− breast cancer (22.7%) in sporadic breast cancers in ethnic Koreans. This was similar to previous ndings that TNBC is associated with a higher incidence of TP53 mutations (80%) than are luminal A breast cancers (12%) [8].
Regarding the clinicopathological implications of these two main breast cancer-associated genes, TP53 mutations in primary tumor tissues were associated with p53 positivity (p < 0.001) and a higher Ki-67 index (p = 0.004), as expected. TP53-mutated breast cancers also had a higher tumor grade (p = 0.008), whereas PIK3CA-mutated breast cancers exhibited more lymphovascular invasion (p = 0.008).
All TP53 and PIK3CA mutations were somatic mutations and were categorized as pathogenic or likely pathogenic variants. In addition, AKT1, ALK, BRCA1, ERBB2, and PTEN were somatically mutated genes and existed without additional pathogenic or likely pathogenic mutations. AR, KIT, and STK11 were also somatically mutated genes, with co-existing pathogenic mutations. The other mutations were only VUS or germline mutations. All three BRCA2 mutations were germline mutations, accompanied by additional pathogenic or likely pathogenic somatic mutations, and were found only in HR+/HER2− breast cancers. Four BRCA1 mutations were found in both groups, one showed LOH of a germline mutation in TNBC and another was a somatic mutation in HR+/HER2− breast cancer. Although BRCA1/2 mutations are strongly associated with TNBC and a high proportion of mutation carriers can develop TNBC, germline BRCA1/2 mutations account for only 15.4% of TNBC [17,18]. In this study, only two (16.7%) BRCA1 mutations were found in sporadic TNBC, and one of them was a germline mutation with LOH.
Both the TNBC and HR+/HER2− groups had no signi cant variants of the ESR1 gene, which are generally found in advanced breast cancer patients treated with endocrine therapy [19]. We monitored patients for a median of 43.5 (range 10-50) months. The follow-up period was short, although recurrences or metastases of TNBC are known to occur within 2-3 years. We had a limited ability to identify the relationship between gene mutations and prognosis because the study subjects had relatively early breast cancer diagnosed as pT2N0-1M0. Recent studies have revealed the relationship between gene mutations and prognosis through analyses of circulating tumor DNA (ctDNA) in advanced breast cancers [20][21][22] Therefore, we analyzed circulating cell-free nucleic acids in preserved blood plasma with the therascreen® PIK3CA RGQ PCR kit (QIAGEN). Circulating cell-free PIK3CA mutations were absent in either groups, which might have been due to the relatively early stage of disease because this diagnostic assay is approved only for advanced disease. We think that the usefulness of therascreen® PIK3CA mutation detection with cell-free nucleic acids at the early stage is still questionable.
Somatic mutations of TP53, PIK3CA, and PTEN are considered founder mutations in primary breast cancers and additional mutations occur in genes involved in other pathways, such as those controlling cell shape and the cytoskeleton [23]. In TNBC, TP53 mutations and methylation of the BRCA1 promoter region are early oncogenic events, and additional structural and epigenetic events occur during clonal progression, resulting in the ampli cation of oncogenes such as MYC, NOTCH2, and NOTCH3, the hampering of tumor suppressor genes, including RB1, PTEN, and KMT2C, and the enhancement of the TGFA oncogene [24]. In this study, the TP53 mutation was predominant in TNBC, and the tumor size seemed to be proportional to the total number of signi cant mutations in TP53-mutated breast cancer, although the cancers were at the same tumor stage (T2, 2-5 cm). Therefore, we inferred that tumor size re ects clonal progression and diversi cation at earlier tumor stages in TP53-mutated breast cancers.

Conclusion
The TP53 mutation is more frequent in TNBC than in HR+/HER2− cancer. It is associated with a higher tumor grade and greater Ki-67 expression in both groups, and with a larger tumor size in TNBC, but not in HR+/HER2-breast cancer. With TP53 mutation, concomitant mutation numbers are proportional to tumor size, re ecting clonal progression. Breast cancer-associated mutations such as those in TP53 and PIK3CA have different biological implications for the proliferation and clonal diversi cation of these two distinct groups of breast cancer.