Cancer-Associated PIK3CA Mutations in Overgrowth Disorders
Authors: Ralitsa R. Madsen, Bart Vanhaesebroeck, Robert K. Semple
Journal: Trends in Molecular Medicine (2018)

Cancer-associated activating PIK3CA mutations, when occurring in isolation during early development, cause a spectrum of rare disorders characterized by asymmetric, and often severe, excessive tissue growth and malformations. Tissues are not uniformly affected, and, surprisingly, no excess of PIK3CA-associated adult cancers has been described. Evidence that low-dose, repurposed cancer therapies may offer effective precision therapy is beginning to emerge. Mouse models driven by endogenous expression of pathogenic, mosaic PIK3CA alleles only partly recapitulate the disease phenotype. Although it is critical for normal cell growth and survival, the role of PIK3CA in early human development is poorly characterized. PIK3CA is one of the most commonly mutated genes in solid cancers. PIK3CA mutations are also found in benign overgrowth syndromes, collectively known as PIK3CA-related overgrowth spectrum (PROS). As in cancer, PIK3CA mutations in PROS arise postzygotically, but unlike in cancer, these mutations arise during embryonic development, with their timing and location critically influencing the resulting disease phenotype. Recent evidence indicates that phosphoinositide 3-kinase (PI3K) pathway inhibitors undergoing trials in cancer can provide a therapy for PROS. Conversely, PROS highlights gaps in our understanding of PI3K's role during embryogenesis and in cancer development. Here, we summarize current knowledge of PROS, evaluate challenges and strategies for disease modeling, and consider the implications of PROS as a paradigm for understanding activating PIK3CA mutations in human development and cancer. PIK3CA is one of the most commonly mutated genes in solid cancers. PIK3CA mutations are also found in benign overgrowth syndromes, collectively known as PIK3CA-related overgrowth spectrum (PROS). As in cancer, PIK3CA mutations in PROS arise postzygotically, but unlike in cancer, these mutations arise during embryonic development, with their timing and location critically influencing the resulting disease phenotype. Recent evidence indicates that phosphoinositide 3-kinase (PI3K) pathway inhibitors undergoing trials in cancer can provide a therapy for PROS. Conversely, PROS highlights gaps in our understanding of PI3K's role during embryogenesis and in cancer development. Here, we summarize current knowledge of PROS, evaluate challenges and strategies for disease modeling, and consider the implications of PROS as a paradigm for understanding activating PIK3CA mutations in human development and cancer. Thirty years ago this year, in 1988, the enzyme phosphoinositide 3-kinase (PI3K) was identified as a signal transducer downstream of activated cell surface growth factor receptors [1Whitman M. et al.Type I phosphatidylinositol kinase makes a novel inositol phospholipid, phosphatidylinositol-3-phosphate.Nature. 1988; 332: 644-646Crossref PubMed Scopus (738) Google Scholar]. Its initial identification in the context of a viral oncogene immediately implicated the PI3K pathway in cancer, and we now know that the genes encoding the p110α catalytic PI3K subunit and its negative regulator, phosphatase and tensin homolog (PTEN), are among the most commonly mutated in solid tumors. It has long been known that heterozygous mutations in PTEN are also responsible for rare, cancer-prone syndromes collectively known as PTEN hamartoma tumor syndrome (PHTS; see Glossary) [2Hollander M.C. et al.PTEN loss in the continuum of common cancers, rare syndromes and mouse models.Nat. Rev. Cancer. 2011; 11: 289-301Crossref PubMed Scopus (351) Google Scholar]. It is only recently, however, that we have learned of rare, but generally benign overgrowth syndromes caused by postzygotic activating mutations in PIK3CA, the gene encoding p110α [3Keppler-Noreuil K.M. et al.Clinical delineation and natural history of the PIK3CA-related overgrowth spectrum.Am. J. Med. Genet. A. 2014; 164: 1713-1733Crossref Scopus (191) Google Scholar]. Collectively known as PIK3CA-related overgrowth spectrum (PROS), these disorders differ from PHTS in important respects: PTEN mutations in PHTS are usually found in all cells, most commonly due to germline transmission, while PIK3CA mutations in PROS occur in mosaic form, are disproportionately found in some tissues, and appear not to be compatible with germline transmission. A key phenotypic difference lies in the increased risk of adult PI3K-associated cancer in PHTS but not PROS. Additional phenotypic variability in PROS arises from the mosaic nature of the disease and complicates efforts to establish experimental models. Nevertheless, such models are critically needed for a better understanding of this rare disorder and for preclinical testing of targeted therapies. Study of rare diseases often improves understanding of common disorders and of fundamental biological mechanisms. Given the critical physiological role of p110α in development and growth, and its frequent pathological hyperactivation in cancer, this is potentially true for PROS, too. Conversely, candidate cancer therapeutics targeting the PI3K pathway bring hope for much needed targeted therapies for PROS, as recently demonstrated in an uncontrolled case series treated with the p110α-specific inhibitor Alpelisib (BYL719) [4Venot Q. et al.Targeted therapy in patients with PIK3CA-related overgrowth syndrome.Nature. 2018; 558: 540-546Crossref PubMed Scopus (247) Google Scholar]. Increased awareness of PROS thus seems timely. This review summarizes current knowledge of p110α activation in PROS, outlines key unanswered questions, and discusses challenges and opportunities in disease modeling and evaluation of novel therapies. The PI3K enzyme first identified was the prototype of what is now known as Class I PI3Ks, which catalyze conversion of the membrane lipid phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] to the second messenger PI(3,4,5)P3 (also known as PIP3). Class I PI3K catalytic subunits are divided into two subclasses – IA and IB – based on differential usage of regulatory subunits. Class IA PI3Ks are heterodimers of one of three catalytic subunits (the PIK3CA gene product p110α, the PIK3CB product p110β, or the PIK3CD product p110δ), tightly bound to one of five regulatory subunits (the PIK3R1 gene products p85α/p55α/p50α, the PIK3R2 product p85β, or the PIK3R3 product p55γ; Figure 1). p110α and p110β are widely expressed, with p110δ predominantly found in leukocytes [5Thorpe L.M. et al.PI3K in cancer: divergent roles of isoforms, modes of activation and therapeutic targeting.Nat. Rev. Cancer. 2015; 15: 7-24Crossref PubMed Scopus (881) Google Scholar]. p110α signals downstream of plasma membrane-associated tyrosine kinases via recruitment of the p85 subunit to tyrosine-phosphorylated receptors/receptor-associated adaptor proteins, or by direct binding to the RAS oncogene [5Thorpe L.M. et al.PI3K in cancer: divergent roles of isoforms, modes of activation and therapeutic targeting.Nat. Rev. Cancer. 2015; 15: 7-24Crossref PubMed Scopus (881) Google Scholar]. p110α activation leads to acute increases in PIP3 and its degradation product PI(3,4)P2, stimulating membrane recruitment of effector proteins with PIP3/PI(3,4)P2-binding domains such as the pleckstrin homology domain. Protein kinase B (AKT) serine/threonine kinases are the best studied PI3K effectors, regulating cell growth, metabolism, survival, and proliferation [5Thorpe L.M. et al.PI3K in cancer: divergent roles of isoforms, modes of activation and therapeutic targeting.Nat. Rev. Cancer. 2015; 15: 7-24Crossref PubMed Scopus (881) Google Scholar, 6Manning B.D. Toker A. AKT/PKB signaling: navigating the network.Cell. 2017; 169: 381-405Abstract Full Text Full Text PDF PubMed Scopus (1824) Google Scholar]. PI3K activity is tightly constrained, resulting in transient and localized PIP3 generation. Both negative feedback by downstream pathway components and the activity of several phospholipid phosphatases are involved in signal termination. Most important of the known lipid phosphatases is the tumor suppressor PTEN, which converts PIP3 back to PI(4,5)P2 [5Thorpe L.M. et al.PI3K in cancer: divergent roles of isoforms, modes of activation and therapeutic targeting.Nat. Rev. Cancer. 2015; 15: 7-24Crossref PubMed Scopus (881) Google Scholar]. The importance of exquisite regulation of PI3K signaling is exemplified by the growing number of genetic disorders known to be caused by mutations in pathway components (Figure 1). PI3K activity was linked to pathological cell growth and oncogenesis early after its discovery, but it was not until 2004 that somatic mutations in PIK3CA were reported in cancers [7Samuels Y. et al.High frequency of mutations of the PIK3CA gene in human cancers.Science. 2004; 304: 554Crossref PubMed Scopus (2915) Google Scholar]. Through high-throughput sequencing, genetic hyperactivation of PI3K/AKT signaling has now become recognized as one of the most frequent 'driver' mechanisms in many cancers [5Thorpe L.M. et al.PI3K in cancer: divergent roles of isoforms, modes of activation and therapeutic targeting.Nat. Rev. Cancer. 2015; 15: 7-24Crossref PubMed Scopus (881) Google Scholar]. Pan-cancer analyses by The Cancer Genome Atlas identified PIK3CA and PTEN among the genes most frequently harboring somatic point mutations in more than 12 different solid tumor types, only behind the tumor suppressor gene TP53 [8Kandoth C. et al.Mutational landscape and significance across 12 major cancer types.Nature. 2013; 503: 333-339Crossref PubMed Scopus (2935) Google Scholar, 9Lawrence M.S. et al.Discovery and saturation analysis of cancer genes across 21 tumour types.Nature. 2014; 505: 495-501Crossref PubMed Scopus (2085) Google Scholar]. Cancers with a high prevalence of activating PIK3CA mutations include breast (>30%), endometrial (>30%), bladder (>20%), colorectal carcinoma (>17%), and head and neck squamous cell carcinoma (>15%) [8Kandoth C. et al.Mutational landscape and significance across 12 major cancer types.Nature. 2013; 503: 333-339Crossref PubMed Scopus (2935) Google Scholar, 9Lawrence M.S. et al.Discovery and saturation analysis of cancer genes across 21 tumour types.Nature. 2014; 505: 495-501Crossref PubMed Scopus (2085) Google Scholar, 10Millis S.Z. et al.Landscape of phosphatidylinositol-3-kinase pathway alterations across 19 784 diverse solid tumors.JAMA Oncol. 2016; 2: 1565-1573Crossref PubMed Scopus (158) Google Scholar, 11Zhang Y. et al.A Pan-Cancer proteogenomic atlas of PI3K/AKT/mTOR pathway alterations.Cancer Cell. 2017; 31 (820–832.e3)Abstract Full Text Full Text PDF Scopus (302) Google Scholar]. Cancers may also have gene amplification or overexpression of any p110 isoform, but only p110α is commonly mutationally activated [5Thorpe L.M. et al.PI3K in cancer: divergent roles of isoforms, modes of activation and therapeutic targeting.Nat. Rev. Cancer. 2015; 15: 7-24Crossref PubMed Scopus (881) Google Scholar]. Mutations span the entire p110α protein except the RAS-binding domain. Over 80% of PIK3CA mutations cluster at three sites, or 'hotspots', namely, glutamates (E) 542 and 545 in the helical domain, and histidine (H) 1047 near the C terminus of the kinase domain (Figure 2). These hotspot variants have the most potent effect on enzymatic activation and downstream biological responses (Table S1 and [11Zhang Y. et al.A Pan-Cancer proteogenomic atlas of PI3K/AKT/mTOR pathway alterations.Cancer Cell. 2017; 31 (820–832.e3)Abstract Full Text Full Text PDF Scopus (302) Google Scholar, 12Dogruluk T. et al.Identification of variant-specific functions of PIK3CA by rapid phenotyping of rare mutations.Cancer Res. 2015; 75: 5341-5354Crossref PubMed Scopus (98) Google Scholar]). Although established as cancer drivers, PIK3CA hotspot mutations were also, surprisingly, identified in benign skin lesions known as epidermal nevi and seborrheic keratoses [13Hafner C. et al.Oncogenic PIK3CA mutations occur in epidermal nevi and seborrheic keratoses with a characteristic mutation pattern.Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 13450-13454Crossref PubMed Scopus (172) Google Scholar]. As in cancer, mutations were only found in the lesions, thus representing another example of genetic mosaicism. More recently, postzygotic, mosaic, activating PIK3CA mutations were also identified in several different forms of segmental overgrowth – that is, asymmetric overgrowth affecting only some parts of the body [14Lindhurst M.J. et al.Mosaic overgrowth with fibroadipose hyperplasia is caused by somatic activating mutations in PIK3CA.Nat. Genet. 2012; 44: 928-933Crossref PubMed Scopus (226) Google Scholar, 15Rivière J.-B. et al.De novo germline and postzygotic mutations in AKT3, PIK3R2 and PIK3CA cause a spectrum of related megalencephaly syndromes.Nat. Genet. 2012; 44: 934-940Crossref PubMed Scopus (520) Google Scholar, 16Kurek K.C. et al.Somatic mosaic activating mutations in PIK3CA cause CLOVES syndrome.Am. J. Hum. Genet. 2012; 90: 1108-1115Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar]. Since then, a wide spectrum of such conditions has been attributed to mosaic genetic activation of p110α. Many affected patients have patterns of overgrowth previously labeled as specific syndromes. The resulting fragmented and inconsistently applied nomenclature complicates classification and fails in the face of intermediate syndromes, leading to the proposed designation of a 'PIK3CA-related overgrowth spectrum', or PROS, to capture the disorders under one label and to reflect disease etiology [3Keppler-Noreuil K.M. et al.Clinical delineation and natural history of the PIK3CA-related overgrowth spectrum.Am. J. Med. Genet. A. 2014; 164: 1713-1733Crossref Scopus (191) Google Scholar]. The severity of PROS is highly variable, ranging from localized overgrowth, for example of a digit, to severe, extensive, and life-threatening overgrowth affecting major vessels and/or critical organs (Figure 3). PROS may be conceived of as a highly anatomically variable admixture of overgrown tissues, with vasculature (capillaries, veins and lymphatics) and adipose tissue often most dramatically affected macroscopically. Many other tissues and organs, including bone, brain, peripheral nerves, liver, skeletal and cardiac muscle, may also be affected [3Keppler-Noreuil K.M. et al.Clinical delineation and natural history of the PIK3CA-related overgrowth spectrum.Am. J. Med. Genet. A. 2014; 164: 1713-1733Crossref Scopus (191) Google Scholar, 17Mirzaa G. et al.PIK3CA-associated developmental disorders exhibit distinct classes of mutations with variable expression and tissue distribution.JCI Insight. 2016; 1e87623Crossref PubMed Google Scholar, 18Leiter S.M. et al.Hypoinsulinaemic, hypoketotic hypoglycaemia due to mosaic genetic activation of PI3-kinase.Eur. J. Endocrinol. 2017; 177: 175-186Crossref PubMed Scopus (24) Google Scholar]. Overgrowth manifests at birth, and progressive overgrowth during childhood is the norm. Soft-tissue overgrowth sometimes persists in adult life, but this cannot currently be predicted. Few reports have identified the identity of mutated cells, with genotyping usually performed on whole tissue. However, PIK3CA mutations have been detected in subcultured dermal fibroblasts [14Lindhurst M.J. et al.Mosaic overgrowth with fibroadipose hyperplasia is caused by somatic activating mutations in PIK3CA.Nat. Genet. 2012; 44: 928-933Crossref PubMed Scopus (226) Google Scholar], adipocytes [16Kurek K.C. et al.Somatic mosaic activating mutations in PIK3CA cause CLOVES syndrome.Am. J. Hum. Genet. 2012; 90: 1108-1115Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar], lymphatic endothelial cells [19Boscolo E. et al.AKT hyper-phosphorylation associated with PI3 K mutations in lymphatic endothelial cells from a patient with lymphatic malformation.Angiogenesis. 2015; 18: 151-162Crossref PubMed Scopus (78) Google Scholar, 20Osborn A.J. et al.Activating PIK3CA alleles and lymphangiogenic phenotype of lymphatic endothelial cells isolated from lymphatic malformations.Hum. Mol. Genet. 2015; 24: 926-938Crossref PubMed Scopus (89) Google Scholar], and skin (epithelial) keratinocytes [21Groesser L. et al.FGFR3, PIK3CA and RAS mutations in benign lichenoid keratosis.Br. J. Dermatol. 2012; 166: 784-788Crossref PubMed Scopus (27) Google Scholar]. Hotspot PIK3CA mutations are only very rarely identified in lymphocyte DNA, even when the overall disease burden is extensive [17Mirzaa G. et al.PIK3CA-associated developmental disorders exhibit distinct classes of mutations with variable expression and tissue distribution.JCI Insight. 2016; 1e87623Crossref PubMed Google Scholar, 22Kuentz P. et al.Molecular diagnosis of PIK3CA-related overgrowth spectrum (PROS) in 162 patients and recommendations for genetic testing.Genet. Med. 2017; 19: 989-997Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar]. Available therapy for PROS centers on judicious surgical 'debulking' of affected regions, with procedures often hazardous due to abnormal vascular anatomy. Surgical or radiological blocking of overgrown blood vessels is also important. Current clinical practice and access of patients to services show major geographical variation, and the unmet need for targeted, less disfiguring approaches to therapy is large. The severity of PROS is most dependent on the timing and location of the initiating mutation (Figure 4, Key Figure). The profile of PIK3CA mutations in PROS closely resembles that in cancer (Figure 2), and hotspot mutations have been suggested to be associated with more severe, focal overgrowth, with rarer non-hotspot mutations often causing more widely distributed but milder overgrowth [3Keppler-Noreuil K.M. et al.Clinical delineation and natural history of the PIK3CA-related overgrowth spectrum.Am. J. Med. Genet. A. 2014; 164: 1713-1733Crossref Scopus (191) Google Scholar, 17Mirzaa G. et al.PIK3CA-associated developmental disorders exhibit distinct classes of mutations with variable expression and tissue distribution.JCI Insight. 2016; 1e87623Crossref PubMed Google Scholar, 22Kuentz P. et al.Molecular diagnosis of PIK3CA-related overgrowth spectrum (PROS) in 162 patients and recommendations for genetic testing.Genet. Med. 2017; 19: 989-997Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar]. However, this fails to explain the full range of observed phenotypes, with clinical observations and biochemical studies of non-hotspot mutations suggesting a more graded phenotypic spectrum [12Dogruluk T. et al.Identification of variant-specific functions of PIK3CA by rapid phenotyping of rare mutations.Cancer Res. 2015; 75: 5341-5354Crossref PubMed Scopus (98) Google Scholar, 17Mirzaa G. et al.PIK3CA-associated developmental disorders exhibit distinct classes of mutations with variable expression and tissue distribution.JCI Insight. 2016; 1e87623Crossref PubMed Google Scholar, 22Kuentz P. et al.Molecular diagnosis of PIK3CA-related overgrowth spectrum (PROS) in 162 patients and recommendations for genetic testing.Genet. Med. 2017; 19: 989-997Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar]. The variability arising from different anatomical distributions of mutations means that very large studies will be required to address a possible genotype–phenotype correlation more definitively. Most studies of cancer and PROS have concentrated on cell-autonomous effects of p110α activation on processes such as cell growth, survival, and migration. However, the mutation burden in PROS at sites of overgrowth is commonly less than the 50% expected if all cells in the tissue were heterozygous for the mutation, and overgrown tissue contains multiple cell types of different embryonic origin. This raises the possibility, still to be tested, that PIK3CA mutation-positive cells exert growth-promoting effects on adjacent or distant cells. This could involve cell–cell interactions, paracrine growth factors, exosomes, and/or alterations to the extracellular matrix. The initial mutation driving PROS is assumed to arise stochastically, and therefore the probability of different developmental lineages being affected should reflect the number of cells of each lineage present in the embryo at the time of mutation. PROS, however, exhibits apparent skewing in the pattern of overgrowth among tissues, with mesoderm-derived tissues (e.g., adipose tissue, vasculature, muscle, bone) and neuroectoderm-derived tissues (e.g., brain, cephalic connective tissue) prominently affected macroscopically (Figure 5). There is much less macroscopic involvement of endoderm-derived structures (e.g., pancreas, liver), and little evidence of epithelial overgrowth beyond epidermal nevi and seborrheic keratoses, both of neuroectodermal origin [23Sarnat H.B. Flores-Sarnat L. Genetics of neural crest and neurocutaneous syndromes.Handbook of Clinical Neurology. 1st ed. Elsevier, 2013: 309-314Crossref Scopus (8) Google Scholar]. The extremely low burden of PIK3CA hotspot mutations in blood, in contrast to non-hotspot variants, which are not infrequently detected in many tissues, including blood, is also of note [15Rivière J.-B. et al.De novo germline and postzygotic mutations in AKT3, PIK3R2 and PIK3CA cause a spectrum of related megalencephaly syndromes.Nat. Genet. 2012; 44: 934-940Crossref PubMed Scopus (520) Google Scholar, 17Mirzaa G. et al.PIK3CA-associated developmental disorders exhibit distinct classes of mutations with variable expression and tissue distribution.JCI Insight. 2016; 1e87623Crossref PubMed Google Scholar, 18Leiter S.M. et al.Hypoinsulinaemic, hypoketotic hypoglycaemia due to mosaic genetic activation of PI3-kinase.Eur. J. Endocrinol. 2017; 177: 175-186Crossref PubMed Scopus (24) Google Scholar, 22Kuentz P. et al.Molecular diagnosis of PIK3CA-related overgrowth spectrum (PROS) in 162 patients and recommendations for genetic testing.Genet. Med. 2017; 19: 989-997Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar]. These observations could be accounted for by processes such as skewing of developmental cell fate decisions, lineage-specific positive or negative selection for PIK3CA-mutant cells, and/or alterations of stem cell dynamics. For instance, hotspot PIK3CA variants may lead to lineage-specific cell loss during or after differentiation due to mechanisms such as oncogene-induced senescence, which is known to occur in cells with strong activation of PI3K signaling [24Kim J.-S. et al.Activation of p53-dependent growth suppression in human cells by mutations in PTEN or PIK3CA.Mol. Cell. Biol. 2007; 27: 662-677Crossref PubMed Scopus (128) Google Scholar]. Hyperactivation of PI3K signaling in stem cells may also lead to attenuation or 'exhaustion' of stem cells' regenerative capacity, a phenomenon best studied in hematopoietic stem cells [25Baumgartner C. et al.An ERK-dependent feedback mechanism prevents hematopoietic stem cell exhaustion.Cell Stem Cell. 2018; 22 (879–892.e6)Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar]. Increased PI3K signaling may affect stemness and/or early lineage determination of pluripotent stem cells (PSCs), and even subtle effects could have a major influence on the PROS phenotype. PI3K/AKT signaling contributes to self-renewal and stemness in models of early development [26Yu J.S. Cui W. Proliferation, survival and metabolism: the role of PI3K/AKT/mTOR signalling in pluripotency and cell fate determination.Development. 2016; 143: 3050-3060Crossref PubMed Scopus (598) Google Scholar, 27Yilmaz A. et al.Defining essential genes for human pluripotent stem cells by CRISPR–Cas9 screening in haploid cells.Nat. Cell Biol. 2018; 20: 610-619Crossref PubMed Scopus (62) Google Scholar], but understanding of dose-dependent effects on human cell fate decisions and crosstalk with key 'stemness' pathways is sparse. The development of human stem cell-based models with activating PIK3CA mutations will permit interrogation of the apparent lineage skewing observed in vivo. The potential role of neural crest stem cells in PROS will be of particular interest given their ability to generate neuroectodermal and mesodermal tissue derivatives, corresponding to tissues most commonly overgrown in PROS. It is important to stress that most diagnostic testing is currently undertaken on accessible areas of macroscopic overgrowth rather than on tissues from internal organs, especially if not frankly enlarged, creating an inevitable bias. Apparent tissue-selective overgrowth may also partly reflect nonuniform PIK3CA gene expression, with the pancreas and liver having more than five times lower mRNA expression than arteries, nerves, adipose tissue, uterus, and breast [28Carithers L.J. et al.A novel approach to high-quality postmortem tissue procurement: the GTEx project.Biopreserv. Biobank. 2015; 13: 311-319Crossref PubMed Scopus (444) Google Scholar]. Moreover, tissues also differ in their capacity to expand as part of physiological adaptation. Adipose tissue and vessels, for example, may dramatically grow and later regress in the face of a transient positive energy balance or tissue injury, and this inherent plasticity may be amplified into greater overgrowth by an endogenous trophic stimulus. More systematic tissue sampling will be needed, ideally with single-cell sequencing, to form an unbiased view of PIK3CA mutation distribution in PROS. To date, the only malignancy reported in PROS is Wilms tumor (nephroblastoma), an embryonal pediatric cancer identified in four of around 200 patients with PROS [29Gripp K.W. et al.Nephroblastomatosis or Wilms tumor in a fourth patient with a somatic PIK3CA mutation.Am. J. Med. Genet. A. 2016; 170: 2559-2569Crossref PubMed Scopus (45) Google Scholar]. Thus, although PIK3CA mutations are very common in cancers, none of the cancer types enriched for such mutations (e.g., endometrial and breast) have been reported in PROS. This may reflect distinct mutation tissue distributions in PROS and cancer. Indeed, as mentioned earlier, most macroscopic overgrowth in PROS occurs in mesodermal and neuroectodermal derivatives (Figure 5), while PIK3CA-associated cancers most commonly arise in ectodermal or endodermal epithelia. Neuroectodermal and mesodermal cancers show very low occurrence, if any, of activating PIK3CA mutations [10Millis S.Z. et al.Landscape of phosphatidylinositol-3-kinase pathway alterations across 19 784 diverse solid tumors.JAMA Oncol. 2016; 2: 1565-1573Crossref PubMed Scopus (158) Google Scholar]. It is also important to emphasize that when denoting a cancer-associated mutation, a cancer 'driver' implies that it confers a selective growth advantage at some point during cancer development, and not that it is sufficient for cancer initiation or maintenance [30Stratton M.R. et al.The cancer genome.Nature. 2009; 458: 719-724Crossref PubMed Scopus (2385) Google Scholar]. This is illustrated by murine cancer models which demonstrate that activating Pik3ca mutations usually require cooperating genetic lesions to induce cancer [31Robinson G. et al.Novel mutations target distinct subgroups of medulloblastoma.Nature. 2012; 488: 43-48Crossref PubMed Scopus (621) Google Scholar, 32Green S. et al.PIK3CAH1047R accelerates and enhances KRASG12D-driven lung tumorigenesis.Cancer Res. 2015; 75: 5378-5391Crossref PubMed Scopus (22) Google Scholar, 33Van Keymeulen A. et al.Reactivation of multipotency by oncogenic PIK3CA induces breast tumour heterogeneity.Nature. 2015; 525: 119-123Crossref PubMed Scopus (227) Google Scholar]. The cellular/tissue context of the mutations is also likely to be an important determinant of their effect on cell behavior, with evidence that even within a single tissue such as the mammary gland, the effect of the hotspot variant Pik3ca-H1047R is dictated by the cell of origin [33Van Keymeulen A. et al.Reactivation of multipotency by oncogenic PIK3CA induces breast tumour heterogeneity.Nature. 2015; 525: 119-123Crossref PubMed Scopus (227) Google Scholar, 34Koren S. et al.PIK3CAH1047R induces multipotency and multi-lineage mammary tumours.Nature. 2015; 525: 114-118Crossref PubMed Scopus (214) Google Scholar]. In marked contrast to PROS, PHTS predisposes to several cancers associated with PI3K activation [35De Santis M. et al.PI3K signaling in tissue hyper-proliferation: from overgrowth syndromes to kidney cysts.Cancers (Basel). 2017; 9: 30Crossref Scopus (29) Google Scholar], including breast, endometrial, and colorectal carcinomas. This may reflect expression of the PTEN mutation in all cells, or the fact that PTEN is not specific for p110α-derived PIP3. Instead, it opposes signaling by any Class I PI3K isoform. Moreover, PTEN has nuclear and lipid phosphatase-independent activities [36Shen W.H. et al.Essential role for nuclear PTEN in maintaining chromosomal integrity.Cell. 2007; 128: 157-170Abstract Full Text Full Text PDF PubMed Scopus (782) Google Scholar, 37Song M.S. et al.Nuclear PTEN regulates the APC-CDH1 tumor-suppressive complex in a phosphatase-independent manner.Cell. 2011; 144: 187-199Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar], although their role in the PHTS phenotype is unclear. A further interesting possibility is that different profiles or strengths of aberrant PI3K activation influence cancer risk. Side-by-side comparisons have not been published, but baseline pathway activation by PIK3CA mutations appears higher than that conferred by heterozygous PTEN loss of function, which results in only modestly increased basal PI3K signaling in some contexts [38Horie Y. et al.Hepatocyte-specific Pten deficiency results in steatohepatitis and hepatocellular carcinomas.J. Clin. Invest. 2004; 113: 1774-1783Crossref PubMed Scopus (539) Google Scholar, 39Papa A. et al.Cancer-associated PTEN mutants act in a dominant-negative manner to suppress PTEN protein function.Cell. 2014; 157: 595-610Abstr