- Email: [email protected]
PIK3CA mutation profiling in Vietnamese patients with breast cancer
Meta Gene 25 (2020) 100709
Contents lists available at ScienceDirect
Meta Gene journal homepage: www.elsevier.com/locate/mgene
PIK3CA mutation profiling in Vietnamese patients with breast cancer a
a
b
c,⁎
Linh Dieu Vuong , To Van Ta , Ha Hoang Chu , Van-Long Truong , Quang Ngoc Nguyen
a,⁎
T
a
Pathology and Molecular Biology Center, National Cancer Hospital K, 30 Cau Buou Street, Thanh Tri, Hanoi, Viet Nam National Key Laboratory of Gene Technology, Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Street, Cau Giay, Hanoi, Viet Nam c Department of Smart Food and Drug, College of BNIT, Inje University, Gimhae 50834, Republic of Korea b
ARTICLE INFO
ABSTRACT
Keywords: Breast cancer HR HER2 PIK3CA mutations Triple negative breast cancer
Activating mutations in phosphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA) gene frequently occur in breast cancer, and are important for the survival of patients with breast cancer. Although PIK3CA mutations have been studied widely in many countries, the relationships between PIK3CA mutations and clinicopathological features are still inconsistent. Moreover, information about PIK3CA mutations in Vietnamese breast cancer has not been investigated. In the present study, we identified PIK3CA mutations in 27.2% of 162 human breast tumors, including 31 exon 9 mutations and 13 exon 20 mutations. In addition, PIK3CA mutations were associated with larger tumor size and low Ki67 level. Moreover, the distribution of PIK3CA mutations in the breast cancer subtypes was analyzed. The PIK3CA mutations was not equally distributed among different intrinsic subtypes, in which the rate of mutations in hormone (HR)-positive subtypes was somewhat higher than that in human epidermal growth factor receptor-2 (HER2)-positive subtypes. Overall, this study provides more information about PIK3CA mutations and suggests prognostic and predictive values for breast cancer management in Vietnamese population.
1. Introduction Phosphatidylinositol 3-kinase (PI3K) pathway play important role in regulating various cellular physiologies such as such as cell proliferation and survival, apoptosis, motility, division, and adhesion (Vivanco and Sawyers, 2002). PI3K is a lipid kinase that interacts with transmembrane tyrosine-kinase growth factor receptors and simultaneously activates downstream signaling pathways, including AKT, mammalian target of rapamycin (mTOR), and mitogen-activated protein kinase (MAPK), which in turn (Aoki and Fujishita, 2017; Yang et al., 2019). Aberrant activation of PI3K pathway occurs frequently in human cancer and is observed in approximately 70% of all breast cancers.One of the major mechanisms for PI3K pathway dysregulation in cancer is mutations in the phosphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA) gene encoding the catalytic subunit p110 alpha of PI3K, suggesting that PIK3CA mutations importantly contribute to breast tumorigenesis (Dupont Jensen et al., 2011). PIK3CA mutations are detected in 20–30% in patients with breast cancer.The vast majority, comprising approximately 90% of PIK3CA mutations, occur in two hotspot regions in exon 9 and exon 20, corresponding to helical and kinase domains, respectively (Dupont Jensen et al., 2011; Shimoi et al., 2018). PIK3CA mutations have different ⁎
prognostic impacts between breast cancer subtypes. Several previous studies indicated that PIK3CA mutations might exhibit positive impact in hormone receptor (HR)-positive breast cancer, whereas PIK3CA mutations that accompany human epidermal growth factor receptor-2 (HER2)-positive tumors might be poor prognostic factors (Ellis et al., 2010; Cizkova et al., 2013; Majewski et al., 2015; Seo et al., 2018). Moreover, recent data from clinical trials suggest that PIK3CA mutations are potent predictive biomarkers for responses to PI3K inhibitors as well as endocrine therapies (Martín et al., 2017). Therefore, identifying PIK3CA mutations provides important information for breast cancer management, as prognostic and predictive values. Many studies have investigated the frequency of PIK3CA mutations and the relationships between PIK3CA mutations and clinicopathological factors, prognostic value, or therapeutic outcome of breast cancer (Dirican et al., 2016; Alqahtani et al., 2019; Wu et al., 2019). However, there are inconsistencies in the results of these different studies even those from the same country. Moreover, information about PIK3CA mutations in Vietnamese breast cancer patients has been unknown. They all suggest that a precise assessment of PI3KCA mutation specific to each patient population may contribute to improving disease management. Therefore, in the present study, we analyzed PIK3CA mutation status, and evaluated possible relationships between
Corresponding authors. E-mail addresses: [email protected] (V.-L. Truong), [email protected] (Q.N. Nguyen).
https://doi.org/10.1016/j.mgene.2020.100709 Received 21 March 2020; Accepted 7 April 2020 Available online 08 April 2020 2214-5400/ © 2020 Published by Elsevier B.V.
Meta Gene 25 (2020) 100709
L.D. Vuong, et al.
PIK3CA mutations and clinicopathological features in Vietnamese breast cancer.
Table 1 Clinicopathological characteristics of Vietnamese breast cancer patients. Clinicopathologicalcharacteristics
2. Materials and methods
N
2.1. Patients and specimens
162
%
< 50.8 > 50.8
77 85
47.5 52.5
Histological subtype Invasive ductal carcinoma Others
133 29
82.1 17.9
4 79 56 23
2.5 48.8 34.6
58 104
35.8 64.2
< 5 cm ≥ 5 cm Unknown
121 20 21
74.7 12.3 13.0
Negative Positive
69 93
42.6 57.4
Negative Positive
88 74
54.3 45.7
Negative Positive
95 67
58.6 41.4
< 20% ≥ 20%
41 121
25.3 74.7
Age
A total of 162 formalin-fixed, paraffin-embedded (FFPE) tumors from patients who were diagnosed as breast cancer at National Cancer Hospital K, Vietnam from 2018 to 2019. This study was approved by Ethnics Committee of Hospital K, and written informed consent was obtained from all participant. Patients who had undergone preoperative adjuvant treatment were excluded in this study. Clinical and pathological features were obtained from medical records and pathological reports.
Tumor grade
1 2 3 Unknown
Lymph node metastasis Yes No
2.2. DNA extraction Genomic DNA was isolated from 5 sections of 10 μm thickness of macro-dissected FFPE tissues, containing at least 50% tumor cells, as identified by two independent pathologists based on hematoxylin and eosin staining. A QIAamp DNA FFPE Tissue Kit (Qiagen, Valencia, CA, USA) were used according to the manufacturer's guidelines. DNA concentrations was determined by Nanodrop 2000 spectrophotometer 9 Thermo Fisher Scientific, Inc., Waltham, MA, USA).
Tumor size
ER
PR
2.3. PIK3CA mutation analysis For each sample, presence of PIK3CA mutations in the exon 9 and 20 were detected using a direct sequencing. Sequences of exon 9 and 20 were amplified by polymerase chain reaction (PCR) with specific primer sets. PCR products were electrophoresed in an agarose gel to confirm successful amplifications of exon 9 and 20 products. The PCR products were then sequenced using a Bigdye Terminator Kit (Applied Biosystems, Foster, CA, USA) according to manufacturer's instructions. All mutations were analyzed by 3130 Genetic Analyzer (Applied Biosystems, Foster, CA, USA). Primer list are presented in Supplementary Table 1.
HER2
Ki67
ER, estrogen receptor; PR, progesterone receptor; HER2, human epidermal growth factor receptor-2.
statistically significant.
2.4. Immunohistochemical analysis
3. Results and discussion
Five μm-thick sections were sliced from each FFPE block and attached onto slide glasses. Immunohistochemical staining for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2) was performed using BenchMark XT system (Ventana Medical System, Tucson, Arizona, USA). The results for ER receptor and PR receptor testing cover a range from low to high levels of receptor expression. A positive test is defined as positive staining of greater than or equal to 1% of tumor cell nucleic. A negative test is defined as staining of less than 1% of tumor cell nucleic. Intensity of staining of tumor cell nucleic may be determined by estimation or a method of quantification. For positive test results, the report includes this estimate of the staining intensity over the entire tumor area of the tissue section. Staining intensity is reported as weak (1+), moderate (2+), or strong (3+). The IHC test gives a score of 0 to 3+ that measures the amount of HER2 receptor protein on the surface of cells in a breast cancer tissue sample. If the score is 0 to 1+, it's called “HER2 negative.” If the score is 2+, it's called “borderline.” A score of 3+ is called “HER2 positive.
The clinicopathological features of patients were summarized in Table 1. The median age at diagnosis was 50.8 years ranging from 23 to 82 years. According to histological subtypes, most of tumors (82.1%) were invasive ductal carcinoma. There were 35.8% patients suffering from lymph node metastasis. The majority of tumors (74.7% for each) were smaller than 5 cm in diameter (excepting for 13.0% cases were unknown tumor size), and high Ki67 level. To better understanding of the PIK3CA mutation spectrum in Vietnamese breast cancer, we did mutational analysis in 162 breast tumors. PIK3CA mutations were identified in 44 of 162 (27.2%) breast tumors, similar to the study of Shimoi et al., which reported a mutation frequency of 33% in a set of 309 Japanese patients with breast cancer (Shimoi et al., 2018). Through extensive screening researches on breast cancer, the rate of PIK3CA mutations was found to be appropriately identical with that of previous reports (Saal et al., 2005; Barbareschi et al., 2007). Mutations occurring in two “hot spot” regions in exon 9 and 20, corresponding to the helical and kinase domains of PIK3CA, respectively, account for up to 90% of all PIK3CA mutation identified in breast cancer (Campbell et al., 2004). In this study, we found 31 mutations in exon 9 (including 3 E542G, 20 E545A, 3 E545G, and 5 E545K) and 13 mutations in exon 20 (including 3 H1046R, 7 H1047R, and 3 H1047D). All mutations discovered in this study were missense mutations, which are pertinent to an oncogenic nature of these mutations. Although the role of each mutation specifically influencing the
2.5. Statistical analysis SPSS software (IBM Corporation, New York, NY, USA) was applied for statistical analysis. Fisher's exact test and χ2 test were used to evaluate the associations between PIK3CA mutation and clinicopathological variables properly. A p < .05 value was considered as 2
Meta Gene 25 (2020) 100709
L.D. Vuong, et al.
clinicopathological factors of breast cancer. The correlation of PIK3CA mutations with pathological and biological characteristics of breast cancer is still a matter of controversy. In the present study, we did not observe any relationship between PIK3CA mutation status and age, histological subtype, tumor grade, nodal status, ER and PR status, HER2 expression, or triple negative breast cancer (TNBC) (Table 2). In some, but not all literatures, PIK3CA mutations are found to correlate with clinicopathologial factors such as lower histological grade, lymph node metastasis, ER and PR positivity, HER2 negativity, and non-TNBC (Saal et al., 2005; Buttitta et al., 2006; Li et al., 2006; Gonzalez-Angulo et al., 2009). Our results have produced conflicts with data from few other reports, for an instance, our result indicated an association of PIK3CA mutations with larger tumor size, whereas other studies found a correlation with smaller tumor size (Pérez-Tenorio et al., 2007; Cizkova et al., 2012). However, our findings are also keeping in line with several previous literatures, in which PIK3CA mutation status was frequently observed in tumors with low Ki67 level, and larger tumor size (Li et al., 2006; Barbareschi et al., 2007; López-Knowles et al., 2014). Some of these discrepancies may be related to sample size, ethnicity, and geographic distribution. In addition, these association may be affected by clinicopathological features specific to each examined population, as evident from a previous studythat indicated differences in the relationships between PIK3CA mutations and clinical factors from two different cohorts (Saal et al., 2005). We were interested in evaluating the distribution of PIK3CA mutations among intrinsic breast cancer subtypes related to therapy responsiveness. As shown in Table 3, mutation frequency of PIK3CA was approximately 15–60% in all subtype, and no statistical significance regarding the mutation frequency was observed between the subtypes. The highest mutation frequency was found in HR-positive/HER2-negative subtype (60%) and the lowest in HR-negative/HER2-postive subtype (15.4%). In fact, PIK3CA mutation frequencies vary among different biological subtypes. Up to 40% of hormone receptor (HR)positive breast tumors appeara PIK3CA mutation, while approximately 20–25% of HER2-positive breast cancers harbor a gene mutation (Loibl et al., 2014). Consistently, our results indicated that the mutation frequency in all HR-positive tumors was 37.3%, while in all HER2-positive tumors was 19.1%. PIK3CA mutations have been demonstrated to be good prognostic factors for HR-positive breast cancer subtype (Ellis et al., 2010). In contrast, HER2-positive breast tumors accompany a PIK3CA mutation are particular dependent on the PI3K pathway, and it has been indicated that coexistence of PIK3CA mutation and HER2 positivity was associated with poor prognosis, as evident from reduced response to anti-HER2 therapy and poorer survival (Cizkova et al., 2013; Majewski et al., 2015; Seo et al., 2018). Expanding upon the observation of previous studies (Stemke-Hale et al., 2008; Shimoi et al., 2018), in which PIK3CA mutation frequency in triple negative breast cancer was 8.5–38%, we found that TNBC mutation frequency of our study was 25.6% (11/43), including 18.6% in exon 9 and 7.0% in exon
Table 2 Correlation between PIK3CA mutations and clinicopathological characteristics. Characteristics
PIK3CA mutation
N Age
< 50.8 > 50.8 Histological subtype Invasive ductal carcinoma Others Tumor grade 1 2 3 Unknown Lymph node metastasis Yes No Tumor size < 5 cm ≥ 5 cm Unknown ER Negative Positive PR Negative Positive HER2 Negative Positive Ki67 < 20% ≥ 20%
p-value
Yes
%
No
%
162
44
27.2
118
72.8
77 85
17 27
22.1 31.8
60 58
77.9 68.2
133 29
36 8
27.1 27.6
97 21
72.9 72.4
4 79 56 23
0 16 11 17
0.0 20.3 19.6 73.9
4 63 45 6
100.0 79.7 80.4 26.1
58 104
14 30
24.1 28.8
44 74
75.9 71.2
121 20 21
26 9 9
21.5 45.0 42.9
95 11 12
78.5 55.0 57.1
69 93
19 25
27.5 26.9
50 68
72.5 73.1
88 74
27 17
30.7 23.0
61 57
69.3 77.0
95 67
26 18
27.4 26.9
69 49
72.6 73.1
41 121
16 28
39.0 23.1
25 93
61.0 76.9
0.166 0.955
1.000 0.777 0.957 0.518 0.024
0.926 0.272 0.944 0.048
ER, estrogen receptor; PR, progesterone receptor; HER2, human epidermal growth factor receptor-2.
function and regulation of the kinase activity of PI3K has not been fully investigated, E545K and H1047R mutations have been demonstrated to enhance its kinase activity in different mechanisms (Samuels et al., 2004; Gkeka et al., 2014; Leontiadou et al., 2018). In addition, our result showed that the frequency of exon 9 mutations washigher than that of exon 20 mutations. In contrast, previous studies reported that exon 20 mutations were predominant in breast cancer, while exon 9 mutations were predominant in colorectal cancer (Saal et al., 2005; Shimoi et al., 2018). These suggest that the Vietnamese breast cancer population might have a higher PIK3CA exon 9 mutation frequency than other populations. However, we did not detect all potential Vietnamese specific mutations in different sites of PIK3CA gene and sample size used in this study was relative small. Therefore, further study needs to be done to confirm this hypothesis. Next, we examined correlation between PIK3CA mutations and Table 3 Distribution of PIK3CA mutations in breast cancer subtypes. PIK3CA mutation
N HR+/HER2+ HR+/HER2HR-/HER2+ TNBC P-value
162 42 25 52 43
Exon 9 mutation
Exon 20 mutation
Yes
%
Yes
%
Yes
%
44 10 15 8 11 0.663
27.2 23.8 60.0 15.4 25.6
31 7 10 6 8 0.556
19.1 16.7 40.0 11.5 18.6
13 3 5 2 3 0.287
8.0 7.1 20.0 3.8 7.0
ER, estrogen receptor; PR, progesterone receptor; HER2, human epidermal growth factor receptor; TNBC, triple-negative breast cancer. +, positive; −, negative. 3
Meta Gene 25 (2020) 100709
L.D. Vuong, et al.
20. In addition, several reports have demonstrated that PIK3CA mutations in the helical and kinase domain also exhibits different prognostic significances, in which helical-domain mutation is a poor prognostic factor while kinase-domain mutation is a good prognostic factor (Barbareschi et al., 2007). It has been showed that exon 9 mutations are associated with early recurrences and death, whereas exon 20 mutations had an excellent prognosis without recurrences or death (Barbareschi et al., 2007; Abramson et al., 2014). In conclusion, our results confirm the high frequency of PIK3CA mutations in Vietnamese patients with breast cancer, showing that mutation rate in exon 9 is higher than that in exon 20. PIK3CA mutations have been indicated to correlate with clinicopathological features such as tumor size and Ki67 level. This study also suggest that PIK3CA mutations may be considered as prognostic and predictive markers during breast cancer management.
Cancer Res. 64, 7678–7681. Cizkova, M., et al., 2012. PIK3CA mutation impact on survival in breast cancer patients and in ERα, PR and ERBB2-based subgroups. Breast Cancer Res. 14, R28. Cizkova, M., et al., 2013. Outcome impact of PIK3CA mutations in HER2-positive breast cancer patients treated with trastuzumab. Br. J. Cancer 108, 1807–1809. Dirican, E., et al., 2016. Mutation distributions and clinical correlations of PIK3CA gene mutations in breast cancer. Tumour Biol. 37, 7033–7045. Dupont Jensen, J., et al., 2011. PIK3CA mutations may be discordant between primary and corresponding metastatic disease in breast Cancer. Clin. Cancer Res. 17, 667. Ellis, M.J., et al., 2010. Phosphatidyl-inositol-3-kinase alpha catalytic subunit mutation and response to neoadjuvant endocrine therapy for estrogen receptor positive breast cancer. Breast Cancer Res. Treat. 119, 379–390. Gkeka, P., et al., 2014. Investigating the structure and dynamics of the PIK3CA wild-type and H1047R oncogenic mutant. PLoS Comput. Biol. 10, e1003895. Gonzalez-Angulo, A.M., et al., 2009. Androgen receptor levels and association with PIK3CA mutations and prognosis in breast Cancer. Clin. Cancer Res. 15, 2472. Leontiadou, H., et al., 2018. Insights into the mechanism of the PIK3CA E545K activating mutation using MD simulations. Sci. Rep. 8, 15544. Li, S.Y., et al., 2006. PIK3CA mutations in breast cancer are associated with poor outcome. Breast Cancer Res. Treat. 96, 91–95. Loibl, S., et al., 2014. PIK3CA mutations are associated with lower rates of pathologic complete response to anti–human epidermal growth factor receptor 2 (HER2) therapy in primary HER2-overexpressing breast Cancer. J. Clin. Oncol. 32, 3212–3220. López-Knowles, E., et al., 2014. Relationship of PIK3CA mutation and pathway activity with antiproliferative response to aromatase inhibition. Breast Cancer Res. 16, R68. Majewski, I.J., et al., 2015. PIK3CA mutations are associated with decreased benefit to neoadjuvant human epidermal growth factor receptor 2-targeted therapies in breast cancer. J. Clin. Oncol. 33, 1334–1339. Martín, M., et al., 2017. A randomized adaptive phase II/III study of buparlisib, a panclass I PI3K inhibitor, combined with paclitaxel for the treatment of HER2-advanced breast cancer (BELLE-4). Ann. Oncol. 28, 313–320. Pérez-Tenorio, G., et al., 2007. PIK3CA mutations and PTEN loss correlate with similar prognostic factors and are not mutually exclusive in breast Cancer. Clin. Cancer Res. 13, 3577–3584. Saal, L.H., et al., 2005. PIK3CA mutations correlate with hormone receptors, node metastasis, and ERBB2, and are mutually exclusive with PTEN loss in human breast carcinoma. Cancer Res. 65, 2554. Samuels, Y., et al., 2004. High frequency of mutations of the PIK3CA gene in human cancers. Science 304, 554. Seo, Y., et al., 2018. PIK3CA mutations and neoadjuvant therapy outcome in patients with human epidermal growth factor receptor 2-positive breast cancer: a sequential analysis. J. Breast Cancer 21, 382–390. Shimoi, T., et al., 2018. PIK3CA mutation profiling in patients with breast cancer, using a highly sensitive detection system. Cancer Sci. 109, 2558–2566. Stemke-Hale, K., et al., 2008. An integrative genomic and proteomic analysis of PIK3CA, PTEN, and AKT mutations in breast cancer. Cancer Res. 68, 6084–6091. Vivanco, I., Sawyers, C.L., 2002. The phosphatidylinositol 3-kinase–AKT pathway in human cancer. Nat. Rev. Cancer 2, 489–501. Wu, H., et al., 2019. The distinct clinicopathological and prognostic implications of PIK3CA mutations in breast cancer patients from Central China. Cancer Manag. Res. 11, 1473–1492. Yang, J., et al., 2019. Targeting PI3K in cancer: mechanisms and advances in clinical trials. Mol. Cancer 18, 26.
Funding The authors received no financial support for this project. Declaration of Competing Interest All the authors declare no conflict of interest. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.mgene.2020.100709. References Abramson, V.G., et al., 2014. Characterization of breast cancers with PI3K mutations in an academic practice setting using SNaPshot profiling. Breast Cancer Res. Treat. 145, 389–399. Alqahtani, A., et al., 2019. PIK3CA gene mutations in solid malignancies: association with Clinicopathological parameters and prognosis. Cancers 12, 93. Aoki, M., Fujishita, T., 2017. Oncogenic roles of the PI3K/AKT/mTOR Axis. In: Hunter, E., Bister, K. (Eds.), Viruses, Genes, and Cancer. Springer International Publishing, Cham, pp. 153–189. Barbareschi, M., et al., 2007. Different prognostic roles of mutations in the helical and kinase domains of the PIK3CA gene in breast carcinomas. Clin. Cancer Res. 13, 6064–6069. Buttitta, F., et al., 2006. PIK3CA mutation and histological type in breast carcinoma: high frequency of mutations in lobular carcinoma. J. Pathol. 208, 350–355. Campbell, I.G., et al., 2004. Mutation of the PIK3CA gene in ovarian and breast cancer.
4
Contents lists available at ScienceDirect
Meta Gene journal homepage: www.elsevier.com/locate/mgene
PIK3CA mutation profiling in Vietnamese patients with breast cancer a
a
b
c,⁎
Linh Dieu Vuong , To Van Ta , Ha Hoang Chu , Van-Long Truong , Quang Ngoc Nguyen
a,⁎
T
a
Pathology and Molecular Biology Center, National Cancer Hospital K, 30 Cau Buou Street, Thanh Tri, Hanoi, Viet Nam National Key Laboratory of Gene Technology, Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Street, Cau Giay, Hanoi, Viet Nam c Department of Smart Food and Drug, College of BNIT, Inje University, Gimhae 50834, Republic of Korea b
ARTICLE INFO
ABSTRACT
Keywords: Breast cancer HR HER2 PIK3CA mutations Triple negative breast cancer
Activating mutations in phosphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA) gene frequently occur in breast cancer, and are important for the survival of patients with breast cancer. Although PIK3CA mutations have been studied widely in many countries, the relationships between PIK3CA mutations and clinicopathological features are still inconsistent. Moreover, information about PIK3CA mutations in Vietnamese breast cancer has not been investigated. In the present study, we identified PIK3CA mutations in 27.2% of 162 human breast tumors, including 31 exon 9 mutations and 13 exon 20 mutations. In addition, PIK3CA mutations were associated with larger tumor size and low Ki67 level. Moreover, the distribution of PIK3CA mutations in the breast cancer subtypes was analyzed. The PIK3CA mutations was not equally distributed among different intrinsic subtypes, in which the rate of mutations in hormone (HR)-positive subtypes was somewhat higher than that in human epidermal growth factor receptor-2 (HER2)-positive subtypes. Overall, this study provides more information about PIK3CA mutations and suggests prognostic and predictive values for breast cancer management in Vietnamese population.
1. Introduction Phosphatidylinositol 3-kinase (PI3K) pathway play important role in regulating various cellular physiologies such as such as cell proliferation and survival, apoptosis, motility, division, and adhesion (Vivanco and Sawyers, 2002). PI3K is a lipid kinase that interacts with transmembrane tyrosine-kinase growth factor receptors and simultaneously activates downstream signaling pathways, including AKT, mammalian target of rapamycin (mTOR), and mitogen-activated protein kinase (MAPK), which in turn (Aoki and Fujishita, 2017; Yang et al., 2019). Aberrant activation of PI3K pathway occurs frequently in human cancer and is observed in approximately 70% of all breast cancers.One of the major mechanisms for PI3K pathway dysregulation in cancer is mutations in the phosphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA) gene encoding the catalytic subunit p110 alpha of PI3K, suggesting that PIK3CA mutations importantly contribute to breast tumorigenesis (Dupont Jensen et al., 2011). PIK3CA mutations are detected in 20–30% in patients with breast cancer.The vast majority, comprising approximately 90% of PIK3CA mutations, occur in two hotspot regions in exon 9 and exon 20, corresponding to helical and kinase domains, respectively (Dupont Jensen et al., 2011; Shimoi et al., 2018). PIK3CA mutations have different ⁎
prognostic impacts between breast cancer subtypes. Several previous studies indicated that PIK3CA mutations might exhibit positive impact in hormone receptor (HR)-positive breast cancer, whereas PIK3CA mutations that accompany human epidermal growth factor receptor-2 (HER2)-positive tumors might be poor prognostic factors (Ellis et al., 2010; Cizkova et al., 2013; Majewski et al., 2015; Seo et al., 2018). Moreover, recent data from clinical trials suggest that PIK3CA mutations are potent predictive biomarkers for responses to PI3K inhibitors as well as endocrine therapies (Martín et al., 2017). Therefore, identifying PIK3CA mutations provides important information for breast cancer management, as prognostic and predictive values. Many studies have investigated the frequency of PIK3CA mutations and the relationships between PIK3CA mutations and clinicopathological factors, prognostic value, or therapeutic outcome of breast cancer (Dirican et al., 2016; Alqahtani et al., 2019; Wu et al., 2019). However, there are inconsistencies in the results of these different studies even those from the same country. Moreover, information about PIK3CA mutations in Vietnamese breast cancer patients has been unknown. They all suggest that a precise assessment of PI3KCA mutation specific to each patient population may contribute to improving disease management. Therefore, in the present study, we analyzed PIK3CA mutation status, and evaluated possible relationships between
Corresponding authors. E-mail addresses: [email protected] (V.-L. Truong), [email protected] (Q.N. Nguyen).
https://doi.org/10.1016/j.mgene.2020.100709 Received 21 March 2020; Accepted 7 April 2020 Available online 08 April 2020 2214-5400/ © 2020 Published by Elsevier B.V.
Meta Gene 25 (2020) 100709
L.D. Vuong, et al.
PIK3CA mutations and clinicopathological features in Vietnamese breast cancer.
Table 1 Clinicopathological characteristics of Vietnamese breast cancer patients. Clinicopathologicalcharacteristics
2. Materials and methods
N
2.1. Patients and specimens
162
%
< 50.8 > 50.8
77 85
47.5 52.5
Histological subtype Invasive ductal carcinoma Others
133 29
82.1 17.9
4 79 56 23
2.5 48.8 34.6
58 104
35.8 64.2
< 5 cm ≥ 5 cm Unknown
121 20 21
74.7 12.3 13.0
Negative Positive
69 93
42.6 57.4
Negative Positive
88 74
54.3 45.7
Negative Positive
95 67
58.6 41.4
< 20% ≥ 20%
41 121
25.3 74.7
Age
A total of 162 formalin-fixed, paraffin-embedded (FFPE) tumors from patients who were diagnosed as breast cancer at National Cancer Hospital K, Vietnam from 2018 to 2019. This study was approved by Ethnics Committee of Hospital K, and written informed consent was obtained from all participant. Patients who had undergone preoperative adjuvant treatment were excluded in this study. Clinical and pathological features were obtained from medical records and pathological reports.
Tumor grade
1 2 3 Unknown
Lymph node metastasis Yes No
2.2. DNA extraction Genomic DNA was isolated from 5 sections of 10 μm thickness of macro-dissected FFPE tissues, containing at least 50% tumor cells, as identified by two independent pathologists based on hematoxylin and eosin staining. A QIAamp DNA FFPE Tissue Kit (Qiagen, Valencia, CA, USA) were used according to the manufacturer's guidelines. DNA concentrations was determined by Nanodrop 2000 spectrophotometer 9 Thermo Fisher Scientific, Inc., Waltham, MA, USA).
Tumor size
ER
PR
2.3. PIK3CA mutation analysis For each sample, presence of PIK3CA mutations in the exon 9 and 20 were detected using a direct sequencing. Sequences of exon 9 and 20 were amplified by polymerase chain reaction (PCR) with specific primer sets. PCR products were electrophoresed in an agarose gel to confirm successful amplifications of exon 9 and 20 products. The PCR products were then sequenced using a Bigdye Terminator Kit (Applied Biosystems, Foster, CA, USA) according to manufacturer's instructions. All mutations were analyzed by 3130 Genetic Analyzer (Applied Biosystems, Foster, CA, USA). Primer list are presented in Supplementary Table 1.
HER2
Ki67
ER, estrogen receptor; PR, progesterone receptor; HER2, human epidermal growth factor receptor-2.
statistically significant.
2.4. Immunohistochemical analysis
3. Results and discussion
Five μm-thick sections were sliced from each FFPE block and attached onto slide glasses. Immunohistochemical staining for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2) was performed using BenchMark XT system (Ventana Medical System, Tucson, Arizona, USA). The results for ER receptor and PR receptor testing cover a range from low to high levels of receptor expression. A positive test is defined as positive staining of greater than or equal to 1% of tumor cell nucleic. A negative test is defined as staining of less than 1% of tumor cell nucleic. Intensity of staining of tumor cell nucleic may be determined by estimation or a method of quantification. For positive test results, the report includes this estimate of the staining intensity over the entire tumor area of the tissue section. Staining intensity is reported as weak (1+), moderate (2+), or strong (3+). The IHC test gives a score of 0 to 3+ that measures the amount of HER2 receptor protein on the surface of cells in a breast cancer tissue sample. If the score is 0 to 1+, it's called “HER2 negative.” If the score is 2+, it's called “borderline.” A score of 3+ is called “HER2 positive.
The clinicopathological features of patients were summarized in Table 1. The median age at diagnosis was 50.8 years ranging from 23 to 82 years. According to histological subtypes, most of tumors (82.1%) were invasive ductal carcinoma. There were 35.8% patients suffering from lymph node metastasis. The majority of tumors (74.7% for each) were smaller than 5 cm in diameter (excepting for 13.0% cases were unknown tumor size), and high Ki67 level. To better understanding of the PIK3CA mutation spectrum in Vietnamese breast cancer, we did mutational analysis in 162 breast tumors. PIK3CA mutations were identified in 44 of 162 (27.2%) breast tumors, similar to the study of Shimoi et al., which reported a mutation frequency of 33% in a set of 309 Japanese patients with breast cancer (Shimoi et al., 2018). Through extensive screening researches on breast cancer, the rate of PIK3CA mutations was found to be appropriately identical with that of previous reports (Saal et al., 2005; Barbareschi et al., 2007). Mutations occurring in two “hot spot” regions in exon 9 and 20, corresponding to the helical and kinase domains of PIK3CA, respectively, account for up to 90% of all PIK3CA mutation identified in breast cancer (Campbell et al., 2004). In this study, we found 31 mutations in exon 9 (including 3 E542G, 20 E545A, 3 E545G, and 5 E545K) and 13 mutations in exon 20 (including 3 H1046R, 7 H1047R, and 3 H1047D). All mutations discovered in this study were missense mutations, which are pertinent to an oncogenic nature of these mutations. Although the role of each mutation specifically influencing the
2.5. Statistical analysis SPSS software (IBM Corporation, New York, NY, USA) was applied for statistical analysis. Fisher's exact test and χ2 test were used to evaluate the associations between PIK3CA mutation and clinicopathological variables properly. A p < .05 value was considered as 2
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clinicopathological factors of breast cancer. The correlation of PIK3CA mutations with pathological and biological characteristics of breast cancer is still a matter of controversy. In the present study, we did not observe any relationship between PIK3CA mutation status and age, histological subtype, tumor grade, nodal status, ER and PR status, HER2 expression, or triple negative breast cancer (TNBC) (Table 2). In some, but not all literatures, PIK3CA mutations are found to correlate with clinicopathologial factors such as lower histological grade, lymph node metastasis, ER and PR positivity, HER2 negativity, and non-TNBC (Saal et al., 2005; Buttitta et al., 2006; Li et al., 2006; Gonzalez-Angulo et al., 2009). Our results have produced conflicts with data from few other reports, for an instance, our result indicated an association of PIK3CA mutations with larger tumor size, whereas other studies found a correlation with smaller tumor size (Pérez-Tenorio et al., 2007; Cizkova et al., 2012). However, our findings are also keeping in line with several previous literatures, in which PIK3CA mutation status was frequently observed in tumors with low Ki67 level, and larger tumor size (Li et al., 2006; Barbareschi et al., 2007; López-Knowles et al., 2014). Some of these discrepancies may be related to sample size, ethnicity, and geographic distribution. In addition, these association may be affected by clinicopathological features specific to each examined population, as evident from a previous studythat indicated differences in the relationships between PIK3CA mutations and clinical factors from two different cohorts (Saal et al., 2005). We were interested in evaluating the distribution of PIK3CA mutations among intrinsic breast cancer subtypes related to therapy responsiveness. As shown in Table 3, mutation frequency of PIK3CA was approximately 15–60% in all subtype, and no statistical significance regarding the mutation frequency was observed between the subtypes. The highest mutation frequency was found in HR-positive/HER2-negative subtype (60%) and the lowest in HR-negative/HER2-postive subtype (15.4%). In fact, PIK3CA mutation frequencies vary among different biological subtypes. Up to 40% of hormone receptor (HR)positive breast tumors appeara PIK3CA mutation, while approximately 20–25% of HER2-positive breast cancers harbor a gene mutation (Loibl et al., 2014). Consistently, our results indicated that the mutation frequency in all HR-positive tumors was 37.3%, while in all HER2-positive tumors was 19.1%. PIK3CA mutations have been demonstrated to be good prognostic factors for HR-positive breast cancer subtype (Ellis et al., 2010). In contrast, HER2-positive breast tumors accompany a PIK3CA mutation are particular dependent on the PI3K pathway, and it has been indicated that coexistence of PIK3CA mutation and HER2 positivity was associated with poor prognosis, as evident from reduced response to anti-HER2 therapy and poorer survival (Cizkova et al., 2013; Majewski et al., 2015; Seo et al., 2018). Expanding upon the observation of previous studies (Stemke-Hale et al., 2008; Shimoi et al., 2018), in which PIK3CA mutation frequency in triple negative breast cancer was 8.5–38%, we found that TNBC mutation frequency of our study was 25.6% (11/43), including 18.6% in exon 9 and 7.0% in exon
Table 2 Correlation between PIK3CA mutations and clinicopathological characteristics. Characteristics
PIK3CA mutation
N Age
< 50.8 > 50.8 Histological subtype Invasive ductal carcinoma Others Tumor grade 1 2 3 Unknown Lymph node metastasis Yes No Tumor size < 5 cm ≥ 5 cm Unknown ER Negative Positive PR Negative Positive HER2 Negative Positive Ki67 < 20% ≥ 20%
p-value
Yes
%
No
%
162
44
27.2
118
72.8
77 85
17 27
22.1 31.8
60 58
77.9 68.2
133 29
36 8
27.1 27.6
97 21
72.9 72.4
4 79 56 23
0 16 11 17
0.0 20.3 19.6 73.9
4 63 45 6
100.0 79.7 80.4 26.1
58 104
14 30
24.1 28.8
44 74
75.9 71.2
121 20 21
26 9 9
21.5 45.0 42.9
95 11 12
78.5 55.0 57.1
69 93
19 25
27.5 26.9
50 68
72.5 73.1
88 74
27 17
30.7 23.0
61 57
69.3 77.0
95 67
26 18
27.4 26.9
69 49
72.6 73.1
41 121
16 28
39.0 23.1
25 93
61.0 76.9
0.166 0.955
1.000 0.777 0.957 0.518 0.024
0.926 0.272 0.944 0.048
ER, estrogen receptor; PR, progesterone receptor; HER2, human epidermal growth factor receptor-2.
function and regulation of the kinase activity of PI3K has not been fully investigated, E545K and H1047R mutations have been demonstrated to enhance its kinase activity in different mechanisms (Samuels et al., 2004; Gkeka et al., 2014; Leontiadou et al., 2018). In addition, our result showed that the frequency of exon 9 mutations washigher than that of exon 20 mutations. In contrast, previous studies reported that exon 20 mutations were predominant in breast cancer, while exon 9 mutations were predominant in colorectal cancer (Saal et al., 2005; Shimoi et al., 2018). These suggest that the Vietnamese breast cancer population might have a higher PIK3CA exon 9 mutation frequency than other populations. However, we did not detect all potential Vietnamese specific mutations in different sites of PIK3CA gene and sample size used in this study was relative small. Therefore, further study needs to be done to confirm this hypothesis. Next, we examined correlation between PIK3CA mutations and Table 3 Distribution of PIK3CA mutations in breast cancer subtypes. PIK3CA mutation
N HR+/HER2+ HR+/HER2HR-/HER2+ TNBC P-value
162 42 25 52 43
Exon 9 mutation
Exon 20 mutation
Yes
%
Yes
%
Yes
%
44 10 15 8 11 0.663
27.2 23.8 60.0 15.4 25.6
31 7 10 6 8 0.556
19.1 16.7 40.0 11.5 18.6
13 3 5 2 3 0.287
8.0 7.1 20.0 3.8 7.0
ER, estrogen receptor; PR, progesterone receptor; HER2, human epidermal growth factor receptor; TNBC, triple-negative breast cancer. +, positive; −, negative. 3
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20. In addition, several reports have demonstrated that PIK3CA mutations in the helical and kinase domain also exhibits different prognostic significances, in which helical-domain mutation is a poor prognostic factor while kinase-domain mutation is a good prognostic factor (Barbareschi et al., 2007). It has been showed that exon 9 mutations are associated with early recurrences and death, whereas exon 20 mutations had an excellent prognosis without recurrences or death (Barbareschi et al., 2007; Abramson et al., 2014). In conclusion, our results confirm the high frequency of PIK3CA mutations in Vietnamese patients with breast cancer, showing that mutation rate in exon 9 is higher than that in exon 20. PIK3CA mutations have been indicated to correlate with clinicopathological features such as tumor size and Ki67 level. This study also suggest that PIK3CA mutations may be considered as prognostic and predictive markers during breast cancer management.
Cancer Res. 64, 7678–7681. Cizkova, M., et al., 2012. PIK3CA mutation impact on survival in breast cancer patients and in ERα, PR and ERBB2-based subgroups. Breast Cancer Res. 14, R28. Cizkova, M., et al., 2013. Outcome impact of PIK3CA mutations in HER2-positive breast cancer patients treated with trastuzumab. Br. J. Cancer 108, 1807–1809. Dirican, E., et al., 2016. Mutation distributions and clinical correlations of PIK3CA gene mutations in breast cancer. Tumour Biol. 37, 7033–7045. Dupont Jensen, J., et al., 2011. PIK3CA mutations may be discordant between primary and corresponding metastatic disease in breast Cancer. Clin. Cancer Res. 17, 667. Ellis, M.J., et al., 2010. Phosphatidyl-inositol-3-kinase alpha catalytic subunit mutation and response to neoadjuvant endocrine therapy for estrogen receptor positive breast cancer. Breast Cancer Res. Treat. 119, 379–390. Gkeka, P., et al., 2014. Investigating the structure and dynamics of the PIK3CA wild-type and H1047R oncogenic mutant. PLoS Comput. Biol. 10, e1003895. Gonzalez-Angulo, A.M., et al., 2009. Androgen receptor levels and association with PIK3CA mutations and prognosis in breast Cancer. Clin. Cancer Res. 15, 2472. Leontiadou, H., et al., 2018. Insights into the mechanism of the PIK3CA E545K activating mutation using MD simulations. Sci. Rep. 8, 15544. Li, S.Y., et al., 2006. PIK3CA mutations in breast cancer are associated with poor outcome. Breast Cancer Res. Treat. 96, 91–95. Loibl, S., et al., 2014. PIK3CA mutations are associated with lower rates of pathologic complete response to anti–human epidermal growth factor receptor 2 (HER2) therapy in primary HER2-overexpressing breast Cancer. J. Clin. Oncol. 32, 3212–3220. López-Knowles, E., et al., 2014. Relationship of PIK3CA mutation and pathway activity with antiproliferative response to aromatase inhibition. Breast Cancer Res. 16, R68. Majewski, I.J., et al., 2015. PIK3CA mutations are associated with decreased benefit to neoadjuvant human epidermal growth factor receptor 2-targeted therapies in breast cancer. J. Clin. Oncol. 33, 1334–1339. Martín, M., et al., 2017. A randomized adaptive phase II/III study of buparlisib, a panclass I PI3K inhibitor, combined with paclitaxel for the treatment of HER2-advanced breast cancer (BELLE-4). Ann. Oncol. 28, 313–320. Pérez-Tenorio, G., et al., 2007. PIK3CA mutations and PTEN loss correlate with similar prognostic factors and are not mutually exclusive in breast Cancer. Clin. Cancer Res. 13, 3577–3584. Saal, L.H., et al., 2005. PIK3CA mutations correlate with hormone receptors, node metastasis, and ERBB2, and are mutually exclusive with PTEN loss in human breast carcinoma. Cancer Res. 65, 2554. Samuels, Y., et al., 2004. High frequency of mutations of the PIK3CA gene in human cancers. Science 304, 554. Seo, Y., et al., 2018. PIK3CA mutations and neoadjuvant therapy outcome in patients with human epidermal growth factor receptor 2-positive breast cancer: a sequential analysis. J. Breast Cancer 21, 382–390. Shimoi, T., et al., 2018. PIK3CA mutation profiling in patients with breast cancer, using a highly sensitive detection system. Cancer Sci. 109, 2558–2566. Stemke-Hale, K., et al., 2008. An integrative genomic and proteomic analysis of PIK3CA, PTEN, and AKT mutations in breast cancer. Cancer Res. 68, 6084–6091. Vivanco, I., Sawyers, C.L., 2002. The phosphatidylinositol 3-kinase–AKT pathway in human cancer. Nat. Rev. Cancer 2, 489–501. Wu, H., et al., 2019. The distinct clinicopathological and prognostic implications of PIK3CA mutations in breast cancer patients from Central China. Cancer Manag. Res. 11, 1473–1492. Yang, J., et al., 2019. Targeting PI3K in cancer: mechanisms and advances in clinical trials. Mol. Cancer 18, 26.
Funding The authors received no financial support for this project. Declaration of Competing Interest All the authors declare no conflict of interest. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.mgene.2020.100709. References Abramson, V.G., et al., 2014. Characterization of breast cancers with PI3K mutations in an academic practice setting using SNaPshot profiling. Breast Cancer Res. Treat. 145, 389–399. Alqahtani, A., et al., 2019. PIK3CA gene mutations in solid malignancies: association with Clinicopathological parameters and prognosis. Cancers 12, 93. Aoki, M., Fujishita, T., 2017. Oncogenic roles of the PI3K/AKT/mTOR Axis. In: Hunter, E., Bister, K. (Eds.), Viruses, Genes, and Cancer. Springer International Publishing, Cham, pp. 153–189. Barbareschi, M., et al., 2007. Different prognostic roles of mutations in the helical and kinase domains of the PIK3CA gene in breast carcinomas. Clin. Cancer Res. 13, 6064–6069. Buttitta, F., et al., 2006. PIK3CA mutation and histological type in breast carcinoma: high frequency of mutations in lobular carcinoma. J. Pathol. 208, 350–355. Campbell, I.G., et al., 2004. Mutation of the PIK3CA gene in ovarian and breast cancer.
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