PIK3R1 Mutations Cause Syndromic Insulin Resistance with Lipoatrophy

Short stature, hyperextensibility of joints and/or inguinal hernia, ocular depression, Rieger anomaly, and teething delay (SHORT) syndrome is a developmental disorder with an unknown genetic cause and hallmarks that include insulin resistance and lack of subcutaneous fat. We ascertained two unrelated individuals with SHORT syndrome, hypothesized that the observed phenotype was most likely due to de novo mutations in the same gene, and performed whole-exome sequencing in the two probands and their unaffected parents. We then confirmed our initial observations in four other subjects with SHORT syndrome from three families, as well as 14 unrelated subjects presenting with syndromic insulin resistance and/or generalized lipoatrophy associated with dysmorphic features and growth retardation. Overall, we identified in nine affected individuals from eight families de novo or inherited PIK3R1 mutations, including a mutational hotspot (c.1945C>T [p.Arg649Trp]) present in four families. PIK3R1 encodes the p85α, p55α, and p50α regulatory subunits of class IA phosphatidylinositol 3 kinases (PI3Ks), which are known to play a key role in insulin signaling. Functional data from fibroblasts derived from individuals with PIK3R1 mutations showed severe insulin resistance for both proximal and distal PI3K-dependent signaling. Our findings extend the genetic causes of severe insulin-resistance syndromes and provide important information with respect to the function of PIK3R1 in normal development and its role in human diseases, including growth delay, Rieger anomaly and other ocular affections, insulin resistance, diabetes, paucity of fat, and ovarian cysts.     

SHORT Syndrome with Partial Lipodystrophy Due to Impaired Phosphatidylinositol 3 Kinase Signaling

The phosphatidylinositol 3 kinase (PI3K) pathway regulates fundamental cellular processes such as metabolism, proliferation, and survival. A central component in this pathway is the p85α regulatory subunit, encoded by PIK3R1. Using whole-exome sequencing, we identified a heterozygous PIK3R1 mutation (c.1945C>T [p.Arg649Trp]) in two unrelated families affected by partial lipodystrophy, low body mass index, short stature, progeroid face, and Rieger anomaly (SHORT syndrome). This mutation led to impaired interaction between p85α and IRS-1 and reduced AKT-mediated insulin signaling in fibroblasts from affected subjects and in reconstituted Pik3r1-knockout preadipocytes. Normal PI3K activity is critical for adipose differentiation and insulin signaling; the mutated PIK3R1 therefore provides a unique link among lipodystrophy, growth, and insulin signaling.     

De Novo Nonsense Mutations in KAT6A,a Lysine Acetyl-Transferase Gene,Cause a Syndrome Including Microcephaly and Global Developmental Delay

Chromatin remodeling through histone acetyltransferase (HAT) and histone deactylase (HDAC) enzymes affects fundamental cellular processes including the cell-cycle, cell differentiation, metabolism, and apoptosis. Nonsense mutations in genes that are involved in histone acetylation and deacetylation result in multiple congenital anomalies with most individuals displaying significant developmental delay, microcephaly and dysmorphism. Here, we report a syndrome caused by de novo heterozygous nonsense mutations in KAT6A (a.k.a., MOZ, MYST3) identified by clinical exome sequencing (CES) in four independent families. The same de novo nonsense mutation (c.3385C>T [p.Arg1129^∗]) was observed in three individuals, and the fourth individual had a nearby de novo nonsense mutation (c.3070C>T [p.Arg1024^∗]). Neither of these variants was present in 1,815 in-house exomes or in public databases. Common features among all four probands include primary microcephaly, global developmental delay including profound speech delay, and craniofacial dysmorphism, as well as more varied features such as feeding difficulties, cardiac defects, and ocular anomalies. We further demonstrate that KAT6A mutations result in dysregulation of H3K9 and H3K18 acetylation and altered P53 signaling. Through histone and non-histone acetylation, KAT6A affects multiple cellular processes and illustrates the complex role of acetylation in regulating development and disease.     

De Novo Mutations in CHAMP1 Cause Intellectual Disability with Severe Speech Impairment

CHAMP1 encodes a protein with a function in kinetochore-microtubule attachment and in the regulation of chromosome segregation, both of which are known to be important for neurodevelopment. By trio whole-exome sequencing, we have identified de novo deleterious mutations in CHAMP1 in five unrelated individuals affected by intellectual disability with severe speech impairment, motor developmental delay, muscular hypotonia, and similar dysmorphic features including short philtrum and a tented upper and everted lover lip. In addition to two frameshift and one nonsense mutations, we found an identical nonsense mutation, c.1192C>T (p.Arg398^∗), in two affected individuals. All mutations, if resulting in a stable protein, are predicted to lead to the loss of the functionally important zinc-finger domains in the C terminus of the protein, which regulate CHAMP1 localization to chromosomes and the mitotic spindle, thereby providing a mechanistic understanding for their pathogenicity. We thus establish deleterious de novo mutations in CHAMP1 as a cause of intellectual disability.     

De Novo Loss-of-Function Mutations in SETD5, Encoding a Methyltransferase in a 3p25 Microdeletion Syndrome Critical Region,Cause Intellectual Disability

To identify further Mendelian causes of intellectual disability (ID), we screened a cohort of 996 individuals with ID for variants in 565 known or candidate genes by using a targeted next-generation sequencing approach. Seven loss-of-function (LoF) mutations—four nonsense (c.1195A>T [p.Lys399^∗], c.1333C>T [p.Arg445^∗], c.1866C>G [p.Tyr622^∗], and c.3001C>T [p.Arg1001^∗]) and three frameshift (c.2177_2178del [p.Thr726Asnfs^∗39], c.3771dup [p.Ser1258Glufs^∗65], and c.3856del [p.Ser1286Leufs^∗84])—were identified in SETD5, a gene predicted to encode a methyltransferase. All mutations were compatible with de novo dominant inheritance. The affected individuals had moderate to severe ID with additional variable features of brachycephaly; a prominent high forehead with synophrys or striking full and broad eyebrows; a long, thin, and tubular nose; long, narrow upslanting palpebral fissures; and large, fleshy low-set ears. Skeletal anomalies, including significant leg-length discrepancy, were a frequent finding in two individuals. Congenital heart defects, inguinal hernia, or hypospadias were also reported. Behavioral problems, including obsessive-compulsive disorder, hand flapping with ritualized behavior, and autism, were prominent features. SETD5 lies within the critical interval for 3p25 microdeletion syndrome. The individuals with SETD5 mutations showed phenotypic similarity to those previously reported with a deletion in 3p25, and thus loss of SETD5 might be sufficient to account for many of the clinical features observed in this condition. Our findings add to the growing evidence that mutations in genes encoding methyltransferases regulating histone modification are important causes of ID. This analysis provides sufficient evidence that rare de novo LoF mutations in SETD5 are a relatively frequent (0.7%) cause of ID.     

Activating PIK3CA Mutations Induce an Epidermal Growth Factor Receptor (EGFR)/Extracellular Signal-regulated Kinase (ERK) Paracrine Signaling Axis in Basal-like Breast Cancer

Mutations in PIK3CA, the gene encoding the p110α catalytic subunit of phosphoinositide 3-kinase (PI3K) have been shown to transform human mammary epithelial cells (MECs). These mutations are present in all breast cancer subtypes, including basal-like breast cancer (BLBC). Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified 72 protein expression changes in human basal-like MECs with knock-in E545K or H1047R PIK3CA mutations versus isogenic MECs with wild-type PIK3CA. Several of these were secreted proteins, cell surface receptors or ECM interacting molecules and were required for growth of PIK3CA mutant cells as well as adjacent cells with wild-type PIK3CA. The proteins identified by MS were enriched among human BLBC cell lines and pointed to a PI3K-dependent amphiregulin/EGFR/ERK signaling axis that is activated in BLBC. Proteins induced by PIK3CA mutations correlated with EGFR signaling and reduced relapse-free survival in BLBC. Treatment with EGFR inhibitors reduced growth of PIK3CA mutant BLBC cell lines and murine mammary tumors driven by a PIK3CA mutant transgene, all together suggesting that PIK3CA mutations promote tumor growth in part by inducing protein changes that activate EGFR.PIK3CA¹, the gene encoding the p110α catalytic subunit of phosphatidylinositide-3 kinase (PI3K), is one of the two most frequently mutated genes in breast cancer. Approximately 80% of these mutations occur in two hot spots in the helical domain (E545K, E542K) and in the catalytic domain (H1047R). PIK3CA activating mutations occur in ∼40% of luminal and HER2-enriched breast cancer subtypes and ∼10% of basal-like breast cancer (BLBC) (1). In this last tumor subtype, mutations in PIK3CA are the most frequent activating kinase mutation. Thus, understanding of how PIK3CA mutations operate in BLBC is important for identifying therapeutic targets in this subtype of the disease, which lacks approved targeted therapies.To elucidate mechanisms by which mutant PIK3CA transforms MECs, we used immortalized, nontumorigenic MCF10A cells, which exhibit basal-like gene expression. Although MCF10A cells require growth factors for proliferation (2), heterozygous knock-in of E545K or H1047R PIK3CA mutation allows growth factor-independent proliferation (3). These knock-in PIK3CA mutant MECs provide a robust model in which to study the impact of these mutations without the effects of random insertion and overexpression associated with ectopic gene transduction. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of these cells identified 72 proteins concordantly altered by both PIK3CA mutations. A significant fraction of these were secreted proteins, cell surface receptors or ECM interacting molecules, suggesting PIK3CA mutations induce changes involving communication with the tumor microenvironment. This analysis identified a PI3K-induced amphiregulin (AREG)-EGFR-ERK signaling pathway that was required for growth of PIK3CA-mutant cells as well as adjacent PIK3CA-WT cells. In addition, these protein changes correlated with poor clinical outcome in BLBC. EGFR antagonists inhibited growth of PIK3CA mutant BLBC tumors, suggesting a potential therapeutic strategy for patients with this molecular subtype of breast cancer.     

De Novo GMNN Mutations Cause Autosomal-Dominant Primordial Dwarfism Associated with Meier-Gorlin Syndrome

Meier-Gorlin syndrome (MGS) is a genetically heterogeneous primordial dwarfism syndrome known to be caused by biallelic loss-of-function mutations in one of five genes encoding pre-replication complex proteins: ORC1, ORC4, ORC6, CDT1, and CDC6. Mutations in these genes cause disruption of the origin of DNA replication initiation. To date, only an autosomal-recessive inheritance pattern has been described in individuals with this disorder, with a molecular etiology established in about three-fourths of cases. Here, we report three subjects with MGS and de novo heterozygous mutations in the 5′ end of GMNN, encoding the DNA replication inhibitor geminin. We identified two truncating mutations in exon 2 (the 1^st coding exon), c.16A>T (p.Lys6^∗) and c.35_38delTCAA (p.Ile12Lysfs^∗4), and one missense mutation, c.50A>G (p.Lys17Arg), affecting the second-to-last nucleotide of exon 2 and possibly RNA splicing. Geminin is present during the S, G2, and M phases of the cell cycle and is degraded during the metaphase-anaphase transition by the anaphase-promoting complex (APC), which recognizes the destruction box sequence near the 5′ end of the geminin protein. All three GMNN mutations identified alter sites 5′ to residue Met28 of the protein, which is located within the destruction box. We present data supporting a gain-of-function mechanism, in which the GMNN mutations result in proteins lacking the destruction box and hence increased protein stability and prolonged inhibition of replication leading to autosomal-dominant MGS.     

Mutations in POGLUT1, Encoding Protein O-Glucosyltransferase 1, Cause Autosomal-Dominant Dowling-Degos Disease

Dowling-Degos disease (DDD) is an autosomal-dominant genodermatosis characterized by progressive and disfiguring reticulate hyperpigmentation. We previously identified loss-of-function mutations in KRT5 but were only able to detect pathogenic mutations in fewer than half of our subjects. To identify additional causes of DDD, we performed exome sequencing in five unrelated affected individuals without mutations in KRT5. Data analysis identified three heterozygous mutations from these individuals, all within the same gene. These mutations, namely c.11G>A (p.Trp4^∗), c.652C>T (p.Arg218^∗), and c.798-2A>C, are within POGLUT1, which encodes protein O-glucosyltransferase 1. Further screening of unexplained cases for POGLUT1 identified six additional mutations, as well as two of the above described mutations. Immunohistochemistry of skin biopsies of affected individuals with POGLUT1 mutations showed significantly weaker POGLUT1 staining in comparison to healthy controls with strong localization of POGLUT1 in the upper parts of the epidermis. Immunoblot analysis revealed that translation of either wild-type (WT) POGLUT1 or of the protein carrying the p.Arg279Trp substitution led to the expected size of about 50 kDa, whereas the c.652C>T (p.Arg218^∗) mutation led to translation of a truncated protein of about 30 kDa. Immunofluorescence analysis identified a colocalization of the WT protein with the endoplasmic reticulum and a notable aggregating pattern for the truncated protein. Recently, mutations in POFUT1, which encodes protein O-fucosyltransferase 1, were also reported to be responsible for DDD. Interestingly, both POGLUT1 and POFUT1 are essential regulators of Notch activity. Our results furthermore emphasize the important role of the Notch pathway in pigmentation and keratinocyte morphology.     

Somatic Mutations of PIK3R1 Promote Gliomagenesis

The phosphoinositide 3-kinase (PI3K) pathway is targeted for frequent alteration in glioblastoma (GBM) and is one of the core GBM pathways defined by The Cancer Genome Atlas. Somatic mutations of PIK3R1 are observed in multiple tumor types, but the tumorigenic activity of these mutations has not been demonstrated in GBM. We show here that somatic mutations in the iSH2 domain of PIK3R1 act as oncogenic driver events. Specifically, introduction of a subset of the mutations identified in human GBM, in the nSH2 and iSH2 domains, increases signaling through the PI3K pathway and promotes tumorigenesis of primary normal human astrocytes in an orthotopic xenograft model. Furthermore, we show that cells that are dependent on mutant P85α-mediated PI3K signaling exhibit increased sensitivity to a small molecule inhibitor of AKT. Together, these results suggest that GBM patients whose tumors carry mutant PIK3R1 alleles may benefit from treatment with inhibitors of AKT.     

Dominant Mutations in KAT6A Cause Intellectual Disability with Recognizable Syndromic Features

Through a multi-center collaboration study, we here report six individuals from five unrelated families, with mutations in KAT6A/MOZ detected by whole-exome sequencing. All five different de novo heterozygous truncating mutations were located in the C-terminal transactivation domain of KAT6A: {"type":"entrez-nucleotide","attrs":{"text":"NM_001099412.1","term_id":"150378462","term_text":"NM_001099412.1"}}NM_001099412.1: c.3116_3117 delCT, p.(Ser1039^∗); c.3830_3831insTT, p.(Arg1278Serfs^∗17); c.3879 dupA, p.(Glu1294Argfs^∗19); c.4108G>T p.(Glu1370^∗) and c.4292 dupT, p.(Leu1431Phefs^∗8). An additional subject with a 0.23 MB microdeletion including the entire KAT6A reading frame was identified with genome-wide array comparative genomic hybridization. Finally, by detailed clinical characterization we provide evidence that heterozygous mutations in KAT6A cause a distinct intellectual disability syndrome. The common phenotype includes hypotonia, intellectual disability, early feeding and oromotor difficulties, microcephaly and/or craniosynostosis, and cardiac defects in combination with subtle facial features such as bitemporal narrowing, broad nasal tip, thin upper lip, posteriorly rotated or low-set ears, and microretrognathia. The identification of human subjects complements previous work from mice and zebrafish where knockouts of Kat6a/kat6a lead to developmental defects.     

Somatic Activating PIK3CA Mutations Cause Venous Malformation

Somatic mutations in TEK, the gene encoding endothelial cell tyrosine kinase receptor TIE2, cause more than half of sporadically occurring unifocal venous malformations (VMs). Here, we report that somatic mutations in PIK3CA, the gene encoding the catalytic p110α subunit of PI3K, cause 54% (27 out of 50) of VMs with no detected TEK mutation. The hotspot mutations c.1624G>A, c.1633G>A, and c.3140A>G (p.Glu542Lys, p.Glu545Lys, and p.His1047Arg), frequent in PIK3CA-associated cancers, overgrowth syndromes, and lymphatic malformation (LM), account for >92% of individuals who carry mutations. Like VM-causative mutations in TEK, the PIK3CA mutations cause chronic activation of AKT, dysregulation of certain important angiogenic factors, and abnormal endothelial cell morphology when expressed in human umbilical vein endothelial cells (HUVECs). The p110α-specific inhibitor BYL719 restores all abnormal phenotypes tested, in PIK3CA- as well as TEK-mutant HUVECs, demonstrating that they operate via the same pathogenic pathways. Nevertheless, significant genotype-phenotype correlations in lesion localization and histology are observed between individuals with mutations in PIK3CA versus TEK, pointing to gene-specific effects.     

BMP4 loss-of-function mutations in developmental eye disorders including SHORT syndrome

BMP4 loss-of-function mutations and deletions have been shown to be associated with ocular, digital, and brain anomalies, but due to the paucity of these reports, the full phenotypic spectrum of human BMP4 mutations is not clear. We screened 133 patients with a variety of ocular disorders for BMP4 coding region mutations or genomic deletions. BMP4 deletions were detected in two patients: a patient affected with SHORT syndrome and a patient with anterior segment anomalies along with craniofacial dysmorphism and cognitive impairment. In addition to this, three intragenic BMP4 mutations were identified. A patient with anophthalmia, microphthalmia with sclerocornea, right-sided diaphragmatic hernia, and hydrocephalus was found to have a c.592C>T (p.R198X) nonsense mutation in BMP4. A frameshift mutation, c.171dupC (p.E58RfsX17), was identified in two half-siblings with anophthalmia/microphthalmia, discordant developmental delay/postaxial polydactyly, and poor growth as well as their unaffected mother; one affected sibling carried an additional BMP4 mutation in the second allele, c.362A>G (p.H121R). This is the first report indicating a role for BMP4 in SHORT syndrome, Axenfeld?CRieger malformation, growth delay, macrocephaly, and diaphragmatic hernia. These results significantly expand the number of reported loss-of-function mutations, further support the critical role of BMP4 in ocular development, and provide additional evidence of variable expression/non-penetrance of BMP4 mutations.     

Exome sequencing identifies PDE4D mutations as another cause of acrodysostosis

Acrodysostosis is a rare autosomal-dominant condition characterized by facial dysostosis, severe brachydactyly with cone-shaped epiphyses, and short stature. Moderate intellectual disability and resistance to multiple hormones might also be present. Recently, a recurrent mutation (c.1102C>T [p.Arg368^∗]) in PRKAR1A has been identified in three individuals with acrodysostosis and resistance to multiple hormones. After studying ten unrelated acrodysostosis cases, we report here de novo PRKAR1A mutations in five out of the ten individuals (we found c.1102C>T [p.Arg368^∗] in four of the ten and c.1117T>C [p.Tyr373His] in one of the ten). We performed exome sequencing in two of the five remaining individuals and selected phosphodiesterase 4D (PDE4D) as a candidate gene. PDE4D encodes a class IV cyclic AMP (cAMP)-specific phosphodiesterase that regulates cAMP concentration. Exome analysis detected heterozygous PDE4D mutations (c.673C>A [p.Pro225Thr] and c.677T>C [p.Phe226Ser]) in these two individuals. Screening of PDE4D identified heterozygous mutations (c.568T>G [p.Ser190Ala] and c.1759A>C [p.Thr587Pro]) in two additional acrodysostosis cases. These mutations occurred de novo in all four cases. The four individuals with PDE4D mutations shared common clinical features, namely characteristic midface and nasal hypoplasia and moderate intellectual disability. Metabolic screening was normal in three of these four individuals. However, resistance to parathyroid hormone and thyrotropin was consistently observed in the five cases with PRKAR1A mutations. Finally, our study further supports the key role of the cAMP signaling pathway in skeletogenesis.     

Deciphering the impact of somatic mutations in exon 20 and exon 9 of PIK3CA gene in breast tumors among Indian women through molecular dynamics approach

We examined 25 breast tumor samples for somatic mutations in exon 20 and exon 9 of PIK3CA gene in South Indian population. Genomic DNA was isolated and amplified for PIK3CA gene, followed by direct sequencing of purified polymerase chain reaction products. We identified PI3K3CA mutations in 5 of 25 (20%), including four of the mutations in p.H1047R and one in p.H1047L. Nucleotide base substitution A to G (c.3140A > G) and A to T (c.3140A > T) results in p.H1047R and p.H1047L mutation in exon 20 of PIK3CA gene. We did not observe any mutation in exon 9 of PIK3CA gene. Furthermore, we investigated the effect of mutations on protein structure and function by the combination of sequence and structure-based in silico prediction methods. This determined the underlying relationship between the mutation and its phenotypic effects. Next step, we complemented by molecular dynamics simulation analysis (30 ns) of native and mutant structures that measured the effect of mutation on protein structure. The obtained results support that the application of computational methods helps predict the biological significance of mutations.     

Identification of Mutations in Distinct Regions of p85 Alpha in Urothelial Cancer

Bladder cancers commonly show genetic aberrations in the phosphatidylinositol 3-kinase signaling pathway. Here we have screened for mutations in PIK3R1, which encodes p85α, one of the regulatory subunits of PI3K. Two hundred and sixty-four bladder tumours and 41 bladder tumour cell lines were screened and 18 mutations were detected. Thirteen mutations were in C-terminal domains and are predicted to interfere with the interaction between p85α and p110α. Five mutations were in the BH domain of PIK3R1. This region has been implicated in p110α-independent roles of p85α, such as binding to and altering the activities of PTEN, Rab4 and Rab5. Expression of these mutant BH-p85α forms in mouse embryonic fibroblasts with p85α knockout indicated that all forms, except the truncation mutants, could bind and stabilize p110α but did not increase AKT phosphorylation, suggesting that BH mutations function independently of p110α. In a panel of 44 bladder tumour cell lines, 80% had reduced PIK3R1 mRNA expression relative to normal urothelial cells. This, along with mutation of PIK3R1, may alter BH domain functioning. Our findings suggest that mutant forms of p85α may play an oncogenic role in bladder cancer, not only via loss of ability to regulate p110α but also via altered function of the BH domain.     

Squamousness: Next-generation sequencing reveals shared molecular features across squamous tumor types

In order to gain a better understanding of the underlying biology of squamous cell carcinoma (SCC), we tested the hypothesis that SCC originating from different organs may possess common molecular alterations. SCC samples (N = 361) were examined using clinical-grade targeted next-generation sequencing (NGS). The most frequent SCC tumor types were head and neck, lung, cutaneous, gastrointestinal and gynecologic cancers. The most common gene alterations were TP53 (64.5% of patients), PIK3CA (28.5%), CDKN2A (24.4%), SOX2 (17.7%), and CCND1 (15.8%). By comparing NGS results of our SCC cohort to a non-SCC cohort (N = 277), we found that CDKN2A, SOX2, NOTCH1, TP53, PIK3CA, CCND1, and FBXW7 were significantly more frequently altered, unlike KRAS, which was less frequently altered in SCC specimens (all P < 0.05; multivariable analysis). Therefore, we identified “squamousness” gene signatures (TP53, PIK3CA, CCND1, CDKN2A, SOX2, NOTCH 1, and FBXW7 aberrations, and absence of KRAS alterations) that were significantly more frequent in SCC versus non-SCC histologies. A multivariable co-alteration analysis established 2 SCC subgroups: (i) patients in whom TP53 and cyclin pathway (CDKN2A and CCND1) alterations strongly correlated but in whom PIK3CA aberrations were less frequent; and (ii) patients with PIK3CA alterations in whom TP53 mutations were less frequent (all P ≤ 0 .001, multivariable analysis). In conclusion, we identified a set of 8 genes altered with significantly different frequencies when SCC and non-SCC were compared, suggesting the existence of patterns for “squamousness.” Targeting the PI3K-AKT-mTOR and/or cyclin pathway components in SCC may be warranted.     

An Integrative Analysis of PIK3CA Mutation,PTEN, and INPP4B Expression in Terms of Trastuzumab Efficacy in HER2-Positive Breast Cancer

The phosphoinositide-3-kinase (PI3K) pathway is commonly deregulated in breast cancer through several mechanisms, including PIK3CA mutation and loss of phosphatase and tensin homolog (PTEN) and inositol polyphosphate 4-phosphatase-II (INPP4B). We aimed to evaluate the predictive relevance of these biomarkers to trastuzumab efficacy in HER2-positive disease. We evaluated the effect of trastuzumab in 43 breast cancer patients with HER2-overexpression who received neoadjuvant treatment. PIK3CA mutation was examined by direct sequencing and digital PCR assay, and PIK3CA copy number was assessed by digital PCR assay of pretreatment tissues. PTEN, pAkt, and INPP4B were assessed by immunohistochemistry. Direct sequencing detected mutant DNA in 21% of all patients, but the incidence increased to 49% using digital PCR. The pathological complete response (pCR) rate in patients with PIK3CA mutations was 29% compared with 67% for those without PIK3CA mutations (P = 0.093), when the mutation was defined as positive if the mutant proportion was more than 10% of total genetic content by digital PCR. Low PTEN expression was associated with less pCR compared to high expression (33% versus 72%, P = 0.034). There were no significant associations of PIK3CA copy number, pAKt, or INPP4B with trastuzumab efficacy. In multivariate analysis, activation of the PI3K pathway due to either PIK3CA mutation or low PTEN were related to poorer response to trastuzumab (OR of predictive pCR was 0.11, 95%CI; 0.03–0.48). In conclusion, activating the PI3K pathway is associated with low pCR to trastuzumab-based treatment in HER2-positive breast cancer. Combined analysis of PIK3CA mutation and PTEN expression may serve as critical indicators to identify patients unlikely to respond to trastuzumab.