Review PIK3CA Mutation Assessment in HR+/HER2− Metastatic Breast Cancer: Overview for Oncology Clinical Practice Carmen Criscitiello 1,2,* , Antonio Marra 1,2,3 and Giuseppe Curigliano 1,2 1 Division of Early Drug Development for Innovative Therapies, IEO, European Institute of Oncology IRCCS, Via Ripamonti, 435, 20141 Milan, Italy; [email protected] (A.M.); [email protected] (G.C.) 2 Department of Oncology and Haemato-Oncology, University of Milano, Via Ripamonti, 435, 20141 Milan, Italy 3 Memorial Sloan Kettering Cancer Center, Department of Pathology, 1275 York Avenue, New York, NY 10065, USA * Correspondence: [email protected]; Tel.: +39-0257489599 Abstract: Activation of the PI3K–AKT–mTOR pathway occurs in several human cancers, including hormone receptor (HR)-positive breast cancer (BC) where is associated with resistance to endocrine therapy and disease progression. In BC, the most common PI3K–AKT–mTOR pathway alteration is represented by PIK3CA oncogenic mutations. These mutations can occur throughout several domains of the p110α catalytic subunit, but the majority are found in the helical and kinase domains (exon 9 and 20) that represent the “hotspots”. Considering the central role of the PI3K–AKT–mTOR pathway in HR-positive BC, several inhibitors (both pan-PI3K and isoform-specific) have been developed and tested in clinical trials. Recently, the PI3Kα-selective inhibitor alpelisib was the first PI3K inhibitor approved for clinical use in HR-positive metastatic BC based on the results of the phase III SOLAR-1 trial. Several methods to assess PIK3CA mutational status in tumor samples have been developed and validated, including real-time polymerase chain reaction (PCR), digital Citation: Criscitiello, C.; Marra, A.; Curigliano, G. PIK3CA Mutation droplet PCR (ddPCR), BEAMing assays, Sanger sequencing, and next-generation sequencing (NGS) Assessment in HR+/HER2− panels. Several new challenges will be expected once alpelisib is widely available in a clinical Metastatic Breast Cancer: Overview setting, including the harmonization of testing procedures for the detection of PI3K–AKT–mTOR for Oncology Clinical Practice. J. Mol. pathway alterations. Herein, we provide an overview on PI3K–AKT–mTOR pathway alterations in Pathol. 2021, 2, 42–54. https:// HR-positive BC, discuss their role in determining prognosis and resistance to endocrine therapy and doi.org/10.3390/jmp2010005 highlight practical considerations about diagnostic methods for the detection of PI3K–AKT–mTOR pathway activation status. Academic Editor: Claudio Bellevicine Keywords: PIK3CA; breast cancer; alpelisib; diagnostic; PCR; next-generation sequencing Received: 2 February 2021 Accepted: 7 March 2021 Published: 11 March 2021 1. Introduction Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in The phosphatidylinositol-3-kinase (PI3K)-protein kinase B (AKT)-mammalian target published maps and institutional affil- of rapamycin (mTOR) cascade is one of the major downstream signaling pathways in iations. human cells and is involved in essential cell processes such as metabolism, survival, proliferation, growth, and motility [1]. The PI3K–AKT–mTOR pathway can be triggered by the activation of various tyrosine kinase receptors (TKRs) or G protein-coupled receptors. Mechanistically, PI3K phosphorylates phosphatidylinositol (4,5)-bisphosphate (PIP2) to phosphatidylinositol (3,4,5,)-trisphosphate (PIP3) at the plasma membrane. Consequently, Copyright: © 2021 by the authors. AKT kinases are activated by PIP3 being able to phosphorylate tuberous sclerosis protein Licensee MDPI, Basel, Switzerland. This article is an open access article 1 (TSC1) and TSC2, and thereby dissociate the TSC1–TSC2 complex. The TSC1–TSC2 distributed under the terms and complex negatively regulates the activity of the kinase mTOR. Therefore, AKT activity conditions of the Creative Commons results in the activation of mTOR complex 1 (mTORC1) and ultimately promotes cell Attribution (CC BY) license (https:// growth and proliferation. Notably, mTORC1 is involved in a negative feedback loop that creativecommons.org/licenses/by/ serves to prevent the overactivation of AKT. The cascade is also antagonized mainly by the 4.0/). tumor suppressor phosphatase and tensin homolog (PTEN), which converts PIP3 to PIP2. J. Mol. Pathol. 2021, 2, 42–54. https://doi.org/10.3390/jmp2010005 https://www.mdpi.com/journal/jmp J. Mol. Pathol. 2021, 2, FOR PEER REVIEW 2 J. Mol. Pathol. 2021, 2 43 that serves to prevent the overactivation of AKT. The cascade is also antagonized mainly by the tumor suppressor phosphatase and tensin homolog (PTEN), which converts PIP3 to PIP2. DysregulationDysregulation of of the the PI3K–AKT–mTOR PI3K–AKT–mTOR pathway pathway occurs occurs in in a largea large variety variety of of human human cancerscancers and and has has been been proven proven to to be be implicated implicated in in tumor tumor development development and and progression progression [2 [2].]. SuchSuch dysregulation dysregulation can can derive derive from from different different mechanisms mechanisms that that include include overactivation overactivation of of growthgrowth factor factor receptors, receptors, activating activating mutations mutations in in the the PI3K PI3K subunits, subunits, PTEN PTEN loss loss of of function, function, andand mutations mutations in in other other genesgenes including AKT. AKT. PI PI3K3K is is a aheterodimer heterodimer with with a regulatory a regulatory and anda catalytic a catalytic subunit. subunit. The Thefour four catalytic catalytic isoforms isoforms of class of classI PI3K I PI3Khave different have different tissue tissueexpres- α β expressionsion patterns. patterns. While While the α theand βand (PIK3CB(PIK3CB) isoforms) isoforms are ubiquitously are ubiquitously expressed expressed in human in γ PIK3CG δ PIK3CD humantissues, tissues, the expression the expression of γ (PIK3CG of ( ) and )δ and (PIK3CD( ) isoforms) isoforms is limited is limited to white to white blood blood cells [3]. PI3Kα is a heterodimeric protein complex comprising the catalytic subunit cells [3]. PI3Kα is a heterodimeric protein complex comprising the catalytic subunit p110a p110a (encoded by the PIK3CA gene located on chromosome 3) and the regulatory subunit (encoded by the PIK3CA gene located on chromosome 3) and the regulatory subunit p85a p85a (encoded by the PIK3R1 gene located on chromosome 5). Mechanistically, p110a binds (encoded by the PIK3R1 gene located on chromosome 5). Mechanistically, p110a binds to to the regulatory subunit p85a, which inhibits p110a, and catalyzes the phosphorylation of the regulatory subunit p85a, which inhibits p110a, and catalyzes the phosphorylation of PIP2 to PIP3 [1]. PI3Kα is the most frequently altered PI3K isoform in solid tumors, playing PIP2 to PIP3 [1]. PI3Kα is the most frequently altered PI3K isoform in solid tumors, play- a prominent role in the aberrant activation of the PI3K-AKT-mTOR pathway [4]. ing a prominent role in the aberrant activation of the PI3K-AKT-mTOR pathway [4]. In breast cancer (BC), aberrant activation of this pathway has been well-documented In breast cancer (BC), aberrant activation of this pathway has been well-documented in estrogen receptor (ER)-positive tumors, being associated with resistance to endocrine in estrogen receptor (ER)-positive tumors, being associated with resistance to endocrine therapy and disease progression [5]. Figure1 schematically summarizes the PI3K-AKT- therapy and disease progression [5]. Figure 1 schematically summarizes the PI3K-AKT- mTOR pathway in ER-positive BC. mTOR pathway in ER-positive BC. Figure 1. Phosphatidylinositol-3-kinase (PI3K)-protein kinase B (AKT)-mammalian target of ra- Figure 1. Phosphatidylinositol-3-kinase (PI3K)-protein kinase B (AKT)-mammalian target of ra- pamycin (mTOR) in ER-positive/HER2-negative breast cancer (ER+/HER2− BC). Abbreviations: pamycin (mTOR) in ER-positive/HER2-negative breast cancer (ER+/HER2− BC). Abbreviations: Akt, protein kinase B; B-RAF, murine sarcoma viral oncogene homolog; ER, estrogen receptor; Akt,ERK1/2, protein extracellular-signal kinase B; B-RAF, regulated murine sarcomakinase 1/2; viral GSK3A/B, oncogene glycogen homolog; synthase ER, estrogen kinase-3 receptor;α/β; HR, ERK1/2,hormone extracellular-signal receptor-positive; regulated MEK, Mitogen-activa kinase 1/2; GSK3A/B,ted protein glycogen kinase; mTOR(C), synthase kinase-3mammalianα/β ;target HR, hormoneof rapamycin receptor-positive; (complex); NF MEK,κB, nuclear Mitogen-activated factor κ-light-chain-enhancer protein kinase; mTOR(C), of activated mammalian B cells; PI3K, target ofphosphatidylinositol rapamycin (complex); 3-kinase; NFκB, PIP, nuclear phosphatidylinositol factor κ-light-chain-enhancer phosphate; PTEN, of activated phosphatase B cells; and PI3K, phosphatidylinositoltensin homolog; RTKs, 3-kinase; receptor PIP, tyrosine phosphatidylinositol kinases; SRC, phosphate;rous sarcoma. PTEN, phosphatase and tensin homolog; RTKs, receptor tyrosine kinases; SRC, rous sarcoma. 2 J. Mol. Pathol. 2021, 2 44 Considering the central role of the PI3K pathway in ER-positive BC, several inhibitors have been developed and tested in clinical trials. If the use of pan-PI3K inhibitors, such as buparlisib and pictilisib, may ensure broad activity with a range of molecular drivers, isoform-specific inhibitors, including taselisib and alpelisib, may reduce off-target toxi- city [6–8]. Indeed, high levels of treatment-related adverse events have been one