PIK3CA Mutational Profiling in a Patient Cohort with HR+/HER2- Advanced Metastatic Breast Cancer at a Tertiary Cancer Center

PIK3CA Mutational Profiling in a Patient Cohort with HR+/HER2- Advanced Metastatic Breast Cancer at a Tertiary Cancer Center | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article PIK3CA Mutational Profiling in a Patient Cohort with HR+/HER2- Advanced Metastatic Breast Cancer at a Tertiary Cancer Center OSAMA ALSMADI, Hikmat Abdel-Razeq, Yazan Talab, Hazem Abdulelah, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2699737/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Jan, 2024 Read the published version in Fortune Journal of Health Sciences → Version 1 posted You are reading this latest preprint version Abstract Common PIK3CA gene activating mutations can be found in 20–30% of all breast cancer cases, and regarded as predictive markers for therapeutic response to PI3K inhibitors. The therascreen PIK3CA mutation companion assay and the alpha-specific PI3K inhibitor, Alpelisib, are FDA-approved for selecting and treating patients with advanced PIK3CA-mutated metastatic breast cancer. The main objective behind this report was to investigate the composition and proportion of PIK3CA mutations using a PIK3CA mutation Therascreen RT-PCR assay, in a patient cohort with receptor-positive/HER2-negative (HR+/HER2) metastatic breast cancer, who were diagnosed and treated at King Hussein Cancer Center (KHCC). Patients with PIK3CA-mutated tumors represented 39.4% (91/231) of all patients. Four PIK3CA mutations comprised 86.8% of all PIK3CA mutations; mainly H1047R (33.3%), E545K (20.9%), E542K (24.2%), and H1047L (8.8%). The four main mutations map to the helical and kinase domains of the PIK3CA encoded protein. C420R was found in only one patient, and E545A was found in two patients. Nine of the 91 mutated patients had shown double PIK3CA mutations (9.9%). In conclusion, PIK3CA is frequently mutated in multiple types of cancers at known ‘Hot-spots’, mainly in the kinase and helical modular domains, which was found consistent with our findings. PIK3CA mutational signature in our metastatic breast cancer cohort varied with a 39.4% (91/231) positivity rate. The PIK3CA mutational screening panel did not capture mutations in the remaining 140 (60.6%) cases; these patients may be mutated in other genes related to breast cancer, or in PIK3CA loci not covered by the Therascreen assay. Survival and clinical outcomes in association with PIK3CA mutational profiles shall be addressed in a follow-up investigation for this patients’ cohort. Biological sciences/Cancer/Breast cancer Biological sciences/Cancer Biological sciences/Drug discovery Biological sciences/Genetics Biological sciences/Molecular biology Figures Figure 1 Introduction PIK3CA activating mutations occur in 20–30% of all cancer cases and valued as predictive markers for response to PI3K inhibitors. PIK3CA belongs to the lipid phosphoinositide 3-kinases (PI3Ks), also known as phosphatidylinositol 3-kinases, that are a family of enzymes composed of four classes that drive a range of physiological functions and cellular processes including cell proliferation, growth, survival, motility and metabolism 1 . Class I PI3Ks are composed of a regulatory and a catalytic subunits that heterodimerize to form a functional biomolecule. PIKs are further divided into IA & IB subsets with the former structured by dimerization between the catalytic p110 (alpha, beta or delta) and p85 regulatory subunits 2 . The p85 subunit has two Src-homology 2 (SH2) domains, and one inter-SH2 domain that constitutively binds to the catalytic p110α subunit, keeping it in an inactive state 3 . Under normal biological conditions, p110 activation occurs when the cell surface receptor tyrosine kinase (RTKs) is autophosphorylated upon its specific ligand binding (e.g. growth factor such as insulin), which then recruits and binds to the cytoplasmic p85/p110 heterodimer complex. This molecular interaction between the phosphorylated receptor and the p85/p110 complex frees the p110 catalytic subunit, enabling it to convert the cellular phosphatidylinositol (3,4)-bisphosphate (PIP2) to phosphatidylinositol (3,4,5)‐triphosphate (PIP3) 4 . PIP3, also known as the second messenger, subsequently phosphorylates PDK1 (3-phosphoinositide-dependent protein kinase-1) which in turn activates AKT (also known as protein kinase B; PKB) 5 , 6 . The overall phosphorylation-based signaling cascade promotes cancer cells proliferation and survival through the mTOR pathway 7 – 9 . Activating alterations in the PI3K-Akt signaling pathway are frequently seen in many human cancers, particularly the common types such as breast, colorectal, endometrial and prostate. Activation by RTKs and somatic driver mutations in specific components of the pathway are the two main underlying oncogenic mechanisms. Female breast cancer is the most commonly diagnosed cancer at the global level, accounting for 11.7% of the total cases 10 . This malignancy and other tumor types has been the focus of many investigations that led to the discovery and design of anti-endocrine and targeted therapies that improved cancer overall survival and clinical outcomes 11 . Many therapies have been approved for the treatment of various malignancies by targeting the PI3K pathway using inhibitory drugs such as BYL719 (alpelisib), CAL101 (idelalisib), BAY 80-6946 (copanlisib), the mTOR inhibitors RAD001 (everolimus) and CCI-779 (temsirolimus). Nevertheless, several resistance mechanisms facilitated by tumor related intrinsic adaptive responses through the reactivation of the PI3K pathway had emerged. These mechanisms are underlined by limited treatment responses and tolerance to therapeutic agents, plus other changes in cellular plasticity, which unfortunately resulted in an increased cancer-associated prevalence and mortalities 12 . More than 70% of breast cancers are hormone receptor (HR) positive and human epidermal growth factor receptor 2 (HER2) negative 13 . Approximately 40% of patients with HR-positive, HER2-negative breast cancer, have activating mutations in the gene PIK3CA (gene encoding p110α catalytic subunit) leading to a hyperactivated and dysregulated PI3K pathway 14 . PIK3CA gene is 34 kb length and located within the chromosome 3q26.3 region. It encodes 1,068 amino acids spanning five modular protein functional domains: amino-terminal p85 binding domain, Ras binding domain, C2 domain, helical domain, and the kinase domain 15 . As exon 1 is entirely UTR, many studies have accordingly numbered the exons according to the coding exons (1–20). Subsequently, these studies refer to E545 mutations as being in exon 9 and H1047 mutations in exon 20 16 . In general, patients found with mutated PIK3CA and hormone receptor-positive, HER2-negative advanced breast cancer, have a worse prognosis compared to those patients with wild-type gene 17 . The recent p110α-specific FDA approved PI3K pathway inhibitor and degrader, alpelisib, has captured much of interest for its efficacy against the invasive and metastatic breast cancer in patients harboring PIK3CA mutations. These mutations mainly occur in one of the two hotspot gene domains, specifically p.E542K and p.E545K in exon 9 (α-helicase domain), and p.H1047R/L in exon 20 (kinase domain) 18 . This group of patients in our study were eligible for receiving the second line therapy, alpelisib alone, or as a combination therapy protocol. More recent data by others, revealed alpelisib therapy had enhanced estrogen signaling in HR + breast cancer, raising a flag for its use as the sole therapeutic agent 19 . Alpelisib when used in combination with fulvestrant endocrine treatment agent according to the SOLAR-1 study, had shown synergistic effects and promising results in patients with advanced metastatic breast cancer 20 . Here, we report on the mutational profiling of PIK3CA in a patient cohort diagnosed with metastatic breast cancer with ER+/HER2- profile, who were considered for treatment based on their genetic profiles. Methods Patient samples and characteristics: In this single center retrospective study, FFPE samples from 231 patients with metastatic breast cancer were received by the molecular diagnostic laboratory, to investigate the presence of PIK3CA actionable mutations using the therascreen Qiagen real-time PCR assay kit. All samples were of breast tissue origin, and ER + /HER − . Histopathological and immunohistochemical examination by qualified pathologists had confirmed the primary diagnosis and tumor percentile, in each case. This study (22 KHCC 041) was approved by KHCC Research Council, and the Institutional Review Board (IRB), in accordance with the international scientific and ethical guidelines. Participants’ written informed consent form (ICF) waiver was approved by KHCC IRB for this retrospective report. PIK3CA mutational analysis: PIK3CA mutational analysis was performed using the FDA-approved Qiagen therascreen PIK3CA RGQ real-time qualitative PCR assay Kit, according to the manufacturer instructions. The kit detects 11 mutations in PIK3CA (exon 7: C420R; exon 9: E542K, E545A, E545D, E545G, E545K, Q546E, and Q546R; and exon 20: H1047L, H1047R, and H1047Y) using genomic DNA derived from formalin-fixed, paraffin-embedded (FFPE) breast tumor tissue with ≥ 20% tumor percentile, or ctDNA extracted from plasma taken from patients with breast cancer. Results Tumor contents of the 231 studies samples range was at 20–95%. Participants’ age ranged between 24–86 years, mean age 53.4 years, median age at 53 years, and the mode age was 46 years. All 231 samples originating from metastatic breast cancer FFPE tissues were analyzed for the presence of PIK3CA mutations. Eleven targeted mutations were screened for in this analysis spanning exons 7 (C420R, E542K, E545A, E545D), 9 (E545G, E545K, and Q546E, Q546R, H1047L), and 20 (H1047R and H1047Y). Of the 11 assay-targeted mutations, E542K (22/231) and E545K (19/231) who map to helicase domain, in addition to H1047R (30/231) and H1047L (8/231) that both map to the kinase domain, were the most prevalent mutations amongst the overall 231 assessed samples (Fig. 1 ), which is consistent with the global hotspot mutational rates in PIK3CA 21 . C420R located within the surface loops of C2 domain, that is proposed to be part of the membrane binding region of p110α 22 , was seen just once in one patient, while E545A was seen in two patients. Interestingly, E545K/H1047L and E545K/H1047R double mutations were detected simultaneously in 2 and 7 patients, respectively. Of the 11 mutations covered in the assay, five (E545D, E545G, Q546E, Q546R, and H1047Y) had not been detected in any of the study participants’ samples. Of all participants, 91/231 (39.4%) had at least one mutation detected, while the remaining 140 cases (60.6%) were wild-type (Table 1 ). Tumor percent (20–95%) was neither age related, nor mutation presence related. Furthermore, PIK3CA wild-type samples were unexpectedly associated with higher percentile tumors at ≥ 50%, while presence of mutation was associated with tumors of lower tumor contents, which we find intriguing and needing explanation and further explanation. Table 1 PIK3CA mutations count and percentiles of the positive samples Coding Exon # Amino Acid Change Base Change P110α Functional domain PIK3CA mutated (mutation percent this study, n = 91) PIK3CA mutated (Global, n = 808) % mutated in all patients (this study, (n = 231) % mutated in global patients (n = 1995) 7 C420R 1258 T > C C2 1 (1.1) 22 (2.7) 0.4 1.1 7 E542K 1624 G > A Helicase 22 (24.2) 94 (11.6) 9.5 4.7 7 E545A 1634 A > C Helicase 2 (2.2) 21 (2.6) 0.9 1.1 7 E545D 1635 G > T Helicase 0 9 (1.1) 0 0.5 9 E545G 1634 A > G Helicase 0 14 (1.7) 0 0.7 9 E545K 1633 G > A Helicase 19 (20.9) 163 (20.2) 8.2 8.2 9 Q546E 1636 C > G Helicase 0 2 (0.2) 0 0.1 9 Q546R 1637 A > G Helicase 0 7 (0.9) 0 0.4 20 H1047Y 3139 C > T Kinase 0 11 (1.4) 0 0.6 20 H1047L 3140 A > T Kinase 8 (8.8) 52 (6.4) 3.5 2.6 20 H1047R 3140 A > G Kinase 30 (33) 288 (35.6) 13.0 14.4 9&20 E545K and H1047L 2 (2.2) 0 0.9 0 9&20 E545K and H1047R 7 (7.7) 0 3.0 0 Total Mutated Samples and percentile 91/231 (39.4) this study; 808/1995 (40.5) global study Total Wild-Type samples and percentile 140/231 (60.6) this study; 1187/1995 (59.5) global study Discussion PIK3CA is frequently mutated in multiple types of cancers at known ‘Hot-spots’, mainly in the kinase and helical modular domains. These are thought to occur early and possibly initiate events leading to malignancies like breast cancer 23 . Of notice, there is a noteworthy absence of mutations in both the Ras Binding Domain (RBD) and within the conserved catalytic site of the kinase domain of PIK3CA 24 . Preclinical evidence in model systems suggests PIK3CA mutations in general have activating (gain of function) properties, however domain-specific mutations may be associated with different AKT (and other pathways) phenotypic consequences, that may be cancer type-specific, or in general common in malignancies. PIK3CA somatic mutations rate varies between 0–50% between the different cancer types, and at 35% with respect to breast cancer 25 , 26 . Several studies reported PIK3CA mutations were strongly associated with AKT pathway activation, growth factor-independent cell proliferation, resistance to apoptosis, and increased invasion and cell migration 18 , 24 , 27 , 28 . Kinase domain mutations appear to be linked more strongly with pathway features enabling cell proliferation 25 , 29 , while helical domain mutations are linked more strongly with features enabling metastatic cell migration and dissemination (e.g. RHO GTPases) 29 , 30 . In our patients’ samples, mutations were detected in both domains, suggesting a direct role for the observed phenotypic features underlying breast cancer development and subsequent invasiveness in these patients. Nevertheless, with respect to possible pathogenic mechanisms, clinical outcomes, and survival in our patient cohort, an in-depth analysis is warranted, and this limitation shall be revisited in a further follow-up investigation. Scarce ethnical/population PIK3CA mutational prevalence currently exists at the global level, mainly through clinical trials data at various geographic areas. The largest data set had been recently presented in a poster session at the San Antonio Breast Cancer Symposium 2021 21 . The authors presented data part of the SOLAR-1 clinical trial, where a total of 1995 patients were enrolled from across 4 regions covering Asia, Europe, Latin America, and Middle East, which included 633, 1025, 136, and 200 participants, respectively. In our study, PIK3CA mutational rate of 39.4% was close but distinct from other regional and global studies from Asia (38.2%), Europe (43.3%), Latin America (38.2%), and Middle East (35.0%). The representative Middle Eastern data in particular was on participants from Egypt and Lebanon. Apart from the European data, our Jordanian participants’ mutational prevalence rate is essentially similar to that reported from populations of Asian, Latin America and the Middle East. When combining all 4 continental geographical areas covered by this SOLAR-1 trial study, the overall prevalence (40.5%) was essentially very comparable to our study (39.4%). The double somatic mutations (E545K/H1047L and E545K/H1047R) seen in 9 (9.9%) of positive 91 cases are assumed in act in cis and likely associated with increased PI3K3CA signaling that activate cellular proliferation and tumor growth underlines by a relived p85a subunit catalytic inhibition 31 . These nine patients are predicted to have improved sensitivity to PI3Ka inhibitors, compared with patients only harboring single-hotspot mutations. To this regard, a met-analysis in silico study 32 combining 10 studies with 6338 patients, showed doubly mutated patients were at 4%, which is essentially similar to our study (3.9%), suggesting our observations are essentially in line with the global data. In another clinical trial (SAFIR02) that enrolled 364 HR + , HER2– patients with metastatic breast cancer, PIK3CA mutations were detected in 104 (28.6%) out of these patients, who poorly responded to chemotherapy, and a shorter overall survival 17 . Altogether, our described patients cohort seem to have an overall PIK3CA mutational rates falling within the regional and global rates, including when individual mutations are considered, suggesting the pathogenic mechanisms and therapeutic outcomes for our patient are anticipated to be in line with regional and global data. Nevertheless, it remains to be seen how these projections will pan out in our planed follow-up clinical investigations. Declarations Acknowledgment: We are grateful to the Science Health Education (SHE) Center and Dana-Farber Cancer Institute for their guidance in editing and review of this manuscript. Special thank you to Editor, Elizabeth Hamlin, for her contribution to the finalization of this manuscript. Author contribution: OA and AT conceived the report idea. OA analyzed data and wrote the manuscript. HR, YT and HA clinically characterized the patients. ZS performed the genotyping and tabulated the genetic data. All authors read and approved the manuscript. 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Relationship of PIK3CA mutation and pathway activity with antiproliferative response to aromatase inhibition. Breast Cancer Res. 2014;16(3):R68. doi: 10.1186/BCR3683 . Krygowska AA, Castellano E. PI3K: A Crucial Piece in the RAS Signaling Puzzle. Cold Spring Harb Perspect Med. 2018;8(6). doi: 10.1101/CSHPERSPECT.A031450 . Vasan N, Razavi P, Johnson JL, et al. Double PIK3CA mutations in cis increase oncogenicity and sensitivity to PI3Kα inhibitors. Science. 2019 Nov 8;366(6466):714–723. doi: 10.1126/science.aaw9032 . Martínez-Sáez, O., Chic, N., Pascual, T. et al. Frequency and spectrum of PIK3CA somatic mutations in breast cancer. Breast Cancer Res 22, 45 (2020). doi.org/10.1186/s13058-020-01284-9 . Additional Declarations No competing interests reported. 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Class I PI3Ks are composed of a regulatory and a catalytic subunits that heterodimerize to form a functional biomolecule. PIKs are further divided into IA \u0026amp; IB subsets with the former structured by dimerization between the catalytic p110 (alpha, beta or delta) and p85 regulatory subunits \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The p85 subunit has two Src-homology 2 (SH2) domains, and one inter-SH2 domain that constitutively binds to the catalytic p110α subunit, keeping it in an inactive state \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eUnder normal biological conditions, p110 activation occurs when the cell surface receptor tyrosine kinase (RTKs) is autophosphorylated upon its specific ligand binding (e.g. growth factor such as insulin), which then recruits and binds to the cytoplasmic p85/p110 heterodimer complex. This molecular interaction between the phosphorylated receptor and the p85/p110 complex frees the p110 catalytic subunit, enabling it to convert the cellular phosphatidylinositol (3,4)-bisphosphate (PIP2) to phosphatidylinositol (3,4,5)‐triphosphate (PIP3) \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. PIP3, also known as the second messenger, subsequently phosphorylates PDK1 (3-phosphoinositide-dependent protein kinase-1) which in turn activates AKT (also known as protein kinase B; PKB) \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The overall phosphorylation-based signaling cascade promotes cancer cells proliferation and survival through the mTOR pathway \u003csup\u003e\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eActivating alterations in the PI3K-Akt signaling pathway are frequently seen in many human cancers, particularly the common types such as breast, colorectal, endometrial and prostate. Activation by RTKs and somatic driver mutations in specific components of the pathway are the two main underlying oncogenic mechanisms. Female breast cancer is the most commonly diagnosed cancer at the global level, accounting for 11.7% of the total cases \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. This malignancy and other tumor types has been the focus of many investigations that led to the discovery and design of anti-endocrine and targeted therapies that improved cancer overall survival and clinical outcomes \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Many therapies have been approved for the treatment of various malignancies by targeting the PI3K pathway using inhibitory drugs such as BYL719 (alpelisib), CAL101 (idelalisib), BAY 80-6946 (copanlisib), the mTOR inhibitors RAD001 (everolimus) and CCI-779 (temsirolimus). Nevertheless, several resistance mechanisms facilitated by tumor related intrinsic adaptive responses through the reactivation of the PI3K pathway had emerged. These mechanisms are underlined by limited treatment responses and tolerance to therapeutic agents, plus other changes in cellular plasticity, which unfortunately resulted in an increased cancer-associated prevalence and mortalities \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMore than 70% of breast cancers are hormone receptor (HR) positive and human epidermal growth factor receptor 2 (HER2) negative \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Approximately 40% of patients with HR-positive, HER2-negative breast cancer, have activating mutations in the gene \u003cem\u003ePIK3CA\u003c/em\u003e (gene encoding p110α catalytic subunit) leading to a hyperactivated and dysregulated PI3K pathway \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003ePIK3CA\u003c/em\u003e gene is 34 kb length and located within the chromosome 3q26.3 region. It encodes 1,068 amino acids spanning five modular protein functional domains: amino-terminal p85 binding domain, Ras binding domain, C2 domain, helical domain, and the kinase domain \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. As exon 1 is entirely UTR, many studies have accordingly numbered the exons according to the coding exons (1\u0026ndash;20). Subsequently, these studies refer to E545 mutations as being in exon 9 and H1047 mutations in exon 20 \u003csup\u003e16\u003c/sup\u003e. In general, patients found with mutated \u003cem\u003ePIK3CA\u003c/em\u003e and hormone receptor-positive, HER2-negative advanced breast cancer, have a worse prognosis compared to those patients with wild-type gene \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe recent p110α-specific FDA approved PI3K pathway inhibitor and degrader, alpelisib, has captured much of interest for its efficacy against the invasive and metastatic breast cancer in patients harboring \u003cem\u003ePIK3CA\u003c/em\u003e mutations. These mutations mainly occur in one of the two hotspot gene domains, specifically p.E542K and p.E545K in exon 9 (α-helicase domain), and p.H1047R/L in exon 20 (kinase domain) \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. This group of patients in our study were eligible for receiving the second line therapy, alpelisib alone, or as a combination therapy protocol. More recent data by others, revealed alpelisib therapy had enhanced estrogen signaling in HR\u0026thinsp;+\u0026thinsp;breast cancer, raising a flag for its use as the sole therapeutic agent \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Alpelisib when used in combination with fulvestrant endocrine treatment agent according to the SOLAR-1 study, had shown synergistic effects and promising results in patients with advanced metastatic breast cancer \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Here, we report on the mutational profiling of \u003cem\u003ePIK3CA\u003c/em\u003e in a patient cohort diagnosed with metastatic breast cancer with ER+/HER2- profile, who were considered for treatment based on their genetic profiles.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatient samples and characteristics:\u003c/h2\u003e \u003cp\u003eIn this single center retrospective study, FFPE samples from 231 patients with metastatic breast cancer were received by the molecular diagnostic laboratory, to investigate the presence of \u003cem\u003ePIK3CA\u003c/em\u003e actionable mutations using the therascreen Qiagen real-time PCR assay kit. All samples were of breast tissue origin, and ER\u003csup\u003e+\u003c/sup\u003e/HER\u003csup\u003e\u0026minus;\u003c/sup\u003e. Histopathological and immunohistochemical examination by qualified pathologists had confirmed the primary diagnosis and tumor percentile, in each case. This study (22 KHCC 041) was approved by KHCC Research Council, and the Institutional Review Board (IRB), in accordance with the international scientific and ethical guidelines. Participants\u0026rsquo; written informed consent form (ICF) waiver was approved by KHCC IRB for this retrospective report.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePIK3CA mutational analysis:\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003ePIK3CA\u003c/em\u003e mutational analysis was performed using the FDA-approved Qiagen therascreen PIK3CA RGQ real-time qualitative PCR assay Kit, according to the manufacturer instructions. The kit detects 11 mutations in \u003cem\u003ePIK3CA\u003c/em\u003e (exon 7: C420R; exon 9: E542K, E545A, E545D, E545G, E545K, Q546E, and Q546R; and exon 20: H1047L, H1047R, and H1047Y) using genomic DNA derived from formalin-fixed, paraffin-embedded (FFPE) breast tumor tissue with \u0026ge;\u0026thinsp;20% tumor percentile, or ctDNA extracted from plasma taken from patients with breast cancer.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eTumor contents of the 231 studies samples range was at 20\u0026ndash;95%. Participants\u0026rsquo; age ranged between 24\u0026ndash;86 years, mean age 53.4 years, median age at 53 years, and the mode age was 46 years. All 231 samples originating from metastatic breast cancer FFPE tissues were analyzed for the presence of \u003cem\u003ePIK3CA\u003c/em\u003e mutations. Eleven targeted mutations were screened for in this analysis spanning exons 7 (C420R, E542K, E545A, E545D), 9 (E545G, E545K, and Q546E, Q546R, H1047L), and 20 (H1047R and H1047Y). Of the 11 assay-targeted mutations, E542K (22/231) and E545K (19/231) who map to helicase domain, in addition to H1047R (30/231) and H1047L (8/231) that both map to the kinase domain, were the most prevalent mutations amongst the overall 231 assessed samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), which is consistent with the global hotspot mutational rates in \u003cem\u003ePIK3CA\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. C420R located within the surface loops of C2 domain, that is proposed to be part of the membrane binding region of p110α \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, was seen just once in one patient, while E545A was seen in two patients. Interestingly, E545K/H1047L and E545K/H1047R double mutations were detected simultaneously in 2 and 7 patients, respectively. Of the 11 mutations covered in the assay, five (E545D, E545G, Q546E, Q546R, and H1047Y) had not been detected in any of the study participants\u0026rsquo; samples.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOf all participants, 91/231 (39.4%) had at least one mutation detected, while the remaining 140 cases (60.6%) were wild-type (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Tumor percent (20\u0026ndash;95%) was neither age related, nor mutation presence related. Furthermore, \u003cem\u003ePIK3CA\u003c/em\u003e wild-type samples were unexpectedly associated with higher percentile tumors at \u0026ge;\u0026thinsp;50%, while presence of mutation was associated with tumors of lower tumor contents, which we find intriguing and needing explanation and further explanation.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePIK3CA mutations count and percentiles of the positive samples\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCoding Exon #\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAmino Acid Change\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBase Change\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP110α\u003c/p\u003e \u003cp\u003eFunctional domain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePIK3CA mutated\u003c/p\u003e \u003cp\u003e(mutation percent this study, n\u0026thinsp;=\u0026thinsp;91)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePIK3CA mutated\u003c/p\u003e \u003cp\u003e(Global, n\u0026thinsp;=\u0026thinsp;808)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e% mutated in all patients\u003c/p\u003e \u003cp\u003e(this study, (n\u0026thinsp;=\u0026thinsp;231)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e% mutated in global patients\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;1995)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC420R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1258 T\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1 (1.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e22 (2.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eE542K\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1624 G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHelicase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e22 (24.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e94 (11.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eE545A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1634 A\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHelicase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2 (2.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e21 (2.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eE545D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1635 G\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHelicase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9 (1.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eE545G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1634 A\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHelicase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e14 (1.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eE545K\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1633 G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHelicase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e19 (20.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e163 (20.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e8.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eQ546E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1636 C\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHelicase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2 (0.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eQ546R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1637 A\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHelicase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7 (0.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH1047Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3139 C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKinase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e11 (1.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH1047L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3140 A\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKinase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8 (8.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e52 (6.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH1047R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3140 A\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKinase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e30 (33)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e288 (35.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e13.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e14.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u0026amp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eE545K and H1047L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2 (2.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u0026amp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eE545K and H1047R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7 (7.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal Mutated Samples and percentile\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c8\" namest=\"c5\"\u003e \u003cp\u003e\u003cb\u003e91/231 (39.4) this study; 808/1995 (40.5) global study\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal Wild-Type samples and percentile\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c8\" namest=\"c5\"\u003e \u003cp\u003e\u003cb\u003e140/231 (60.6) this study; 1187/1995 (59.5) global study\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003e \u003cp\u003e \u003cem\u003ePIK3CA\u003c/em\u003e is frequently mutated in multiple types of cancers at known \u0026lsquo;Hot-spots\u0026rsquo;, mainly in the kinase and helical modular domains. These are thought to occur early and possibly initiate events leading to malignancies like breast cancer \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Of notice, there is a noteworthy absence of mutations in both the Ras Binding Domain (RBD) and within the conserved catalytic site of the kinase domain of \u003cem\u003ePIK3CA\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePreclinical evidence in model systems suggests \u003cem\u003ePIK3CA\u003c/em\u003e mutations in general have activating (gain of function) properties, however domain-specific mutations may be associated with different AKT (and other pathways) phenotypic consequences, that may be cancer type-specific, or in general common in malignancies. \u003cem\u003ePIK3CA\u003c/em\u003e somatic mutations rate varies between 0\u0026ndash;50% between the different cancer types, and at 35% with respect to breast cancer \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Several studies reported \u003cem\u003ePIK3CA\u003c/em\u003e mutations were strongly associated with AKT pathway activation, growth factor-independent cell proliferation, resistance to apoptosis, and increased invasion and cell migration \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eKinase domain mutations appear to be linked more strongly with pathway features enabling cell proliferation \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, while helical domain mutations are linked more strongly with features enabling metastatic cell migration and dissemination (e.g. RHO GTPases) \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. In our patients\u0026rsquo; samples, mutations were detected in both domains, suggesting a direct role for the observed phenotypic features underlying breast cancer development and subsequent invasiveness in these patients. Nevertheless, with respect to possible pathogenic mechanisms, clinical outcomes, and survival in our patient cohort, an in-depth analysis is warranted, and this limitation shall be revisited in a further follow-up investigation.\u003c/p\u003e \u003cp\u003eScarce ethnical/population \u003cem\u003ePIK3CA\u003c/em\u003e mutational prevalence currently exists at the global level, mainly through clinical trials data at various geographic areas. The largest data set had been recently presented in a poster session at the San Antonio Breast Cancer Symposium 2021 \u003csup\u003e21\u003c/sup\u003e. The authors presented data part of the SOLAR-1 clinical trial, where a total of 1995 patients were enrolled from across 4 regions covering Asia, Europe, Latin America, and Middle East, which included 633, 1025, 136, and 200 participants, respectively. In our study, \u003cem\u003ePIK3CA\u003c/em\u003e mutational rate of 39.4% was close but distinct from other regional and global studies from Asia (38.2%), Europe (43.3%), Latin America (38.2%), and Middle East (35.0%). The representative Middle Eastern data in particular was on participants from Egypt and Lebanon. Apart from the European data, our Jordanian participants\u0026rsquo; mutational prevalence rate is essentially similar to that reported from populations of Asian, Latin America and the Middle East. When combining all 4 continental geographical areas covered by this SOLAR-1 trial study, the overall prevalence (40.5%) was essentially very comparable to our study (39.4%). The double somatic mutations (E545K/H1047L and E545K/H1047R) seen in 9 (9.9%) of positive 91 cases are assumed in act in cis and likely associated with increased PI3K3CA signaling that activate cellular proliferation and tumor growth underlines by a relived p85a subunit catalytic inhibition \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. These nine patients are predicted to have improved sensitivity to PI3Ka inhibitors, compared with patients only harboring single-hotspot mutations. To this regard, a met-analysis in silico study \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e combining 10 studies with 6338 patients, showed doubly mutated patients were at 4%, which is essentially similar to our study (3.9%), suggesting our observations are essentially in line with the global data.\u003c/p\u003e \u003cp\u003eIn another clinical trial (SAFIR02) that enrolled 364 HR\u003csup\u003e+\u003c/sup\u003e, HER2\u0026ndash; patients with metastatic breast cancer, \u003cem\u003ePIK3CA\u003c/em\u003e mutations were detected in 104 (28.6%) out of these patients, who poorly responded to chemotherapy, and a shorter overall survival \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Altogether, our described patients cohort seem to have an overall \u003cem\u003ePIK3CA\u003c/em\u003e mutational rates falling within the regional and global rates, including when individual mutations are considered, suggesting the pathogenic mechanisms and therapeutic outcomes for our patient are anticipated to be in line with regional and global data. Nevertheless, it remains to be seen how these projections will pan out in our planed follow-up clinical investigations.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgment:\u003c/p\u003e\u003cp\u003e\u003cem\u003eWe are grateful to the Science Health Education (SHE) Center and Dana-Farber Cancer Institute for their guidance in editing and review of this manuscript. Special thank you to Editor, Elizabeth Hamlin, for her contribution to the finalization of this manuscript.\u003c/em\u003e\u003c/p\u003e\u003cp\u003eAuthor contribution:\u003c/p\u003e\u003cp\u003eOA and AT conceived the report idea. OA analyzed data and wrote the manuscript. HR, YT and HA clinically characterized the patients. ZS performed the genotyping and tabulated the genetic data. All authors read and approved the manuscript.\u003c/p\u003e\u003cp\u003eData Availability:\u003c/p\u003e\u003cp\u003eAll data generated or analyzed during this study are included in this article (Table 1).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eVanhaesebroeck B, Stephens L, Hawkins P. PI3K signalling: the path to discovery and understanding. Nat Rev Mol Cell Biol. 2012;13(3):195\u0026ndash;203. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nrm3290\u003c/span\u003e\u003cspan address=\"10.1038/nrm3290\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarpenter CL, Duckworth BC, Auger KR, Cohen B, Schaffhausen BS, Cantley LC. 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Breast Cancer Res 22, 45 (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003edoi.org/10.1186/s13058-020-01284-9\u003c/span\u003e\u003cspan address=\"10.1186/s13058-020-01284-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-2699737/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2699737/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCommon \u003cem\u003ePIK3CA\u003c/em\u003e gene activating mutations can be found in 20–30% of all breast cancer cases, and regarded as predictive markers for therapeutic response to PI3K inhibitors. The therascreen PIK3CA mutation companion assay and the alpha-specific PI3K inhibitor, Alpelisib, are FDA-approved for selecting and treating patients with advanced PIK3CA-mutated metastatic breast cancer. The main objective behind this report was to investigate the composition and proportion of PIK3CA mutations using a \u003cem\u003ePIK3CA\u003c/em\u003e mutation Therascreen RT-PCR assay, in a patient cohort with receptor-positive/HER2-negative (HR+/HER2) metastatic breast cancer, who were diagnosed and treated at King Hussein Cancer Center (KHCC). Patients with PIK3CA-mutated tumors represented 39.4% (91/231) of all patients. Four PIK3CA mutations comprised 86.8% of all \u003cem\u003ePIK3CA\u003c/em\u003e mutations; mainly H1047R (33.3%), E545K (20.9%), E542K (24.2%), and H1047L (8.8%). The four main mutations map to the helical and kinase domains of the PIK3CA encoded protein. C420R was found in only one patient, and E545A was found in two patients. Nine of the 91 mutated patients had shown double \u003cem\u003ePIK3CA\u003c/em\u003e mutations (9.9%). In conclusion, \u003cem\u003ePIK3CA\u003c/em\u003e is frequently mutated in multiple types of cancers at known ‘Hot-spots’, mainly in the kinase and helical modular domains, which was found consistent with our findings. PIK3CA mutational signature in our metastatic breast cancer cohort varied with a 39.4% (91/231) positivity rate. The PIK3CA mutational screening panel did not capture mutations in the remaining 140 (60.6%) cases; these patients may be mutated in other genes related to breast cancer, or in PIK3CA loci not covered by the Therascreen assay. Survival and clinical outcomes in association with \u003cem\u003ePIK3CA\u003c/em\u003e mutational profiles shall be addressed in a follow-up investigation for this patients’ cohort.\u003c/p\u003e","manuscriptTitle":"PIK3CA Mutational Profiling in a Patient Cohort with HR+/HER2- Advanced Metastatic Breast Cancer at a Tertiary Cancer Center","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-03-29 21:52:52","doi":"10.21203/rs.3.rs-2699737/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7576328f-eddf-416e-88cb-781e14197cc9","owner":[],"postedDate":"March 29th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":20229251,"name":"Biological sciences/Cancer/Breast cancer"},{"id":20229252,"name":"Biological sciences/Cancer"},{"id":20229253,"name":"Biological sciences/Drug discovery"},{"id":20229254,"name":"Biological sciences/Genetics"},{"id":20229255,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2024-04-01T06:17:56+00:00","versionOfRecord":{"articleIdentity":"rs-2699737","link":"https://doi.org/10.26502/fjhs.174","journal":{"identity":"fortune-journal-of-health-sciences","isVorOnly":true,"title":"Fortune Journal of Health Sciences"},"publishedOn":"2024-01-01 06:17:56","publishedOnDateReadable":"January 1st, 2024"},"versionCreatedAt":"2023-03-29 21:52:52","video":"","vorDoi":"10.26502/fjhs.174","vorDoiUrl":"https://doi.org/10.26502/fjhs.174","workflowStages":[]},"version":"v1","identity":"rs-2699737","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2699737","identity":"rs-2699737","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}