Research

JAMA Oncology | Original Investigation

Landscape of Phosphatidylinositol-3-Kinase Pathway

Alterations Across 19 784 Diverse Solid Tumors
Sherri Z. Millis, PhD; Sadakatsu Ikeda, MD; Sandeep Reddy, MD; Zoran Gatalica, MD; Razelle Kurzrock, MD

Editorial page 1543

IMPORTANCE Molecular aberrations in the phosphatidylinositol-3-kinase (PI3K) pathway Supplemental content at
drive tumorigenesis. Frequently co-occurring alterations in hormone receptors and/or human jamaoncology.com
epidermal growth factor receptor 2 (HER2) may be relevant to mechanisms of response and
resistance.

OBJECTIVE To identify patterns of aberration in the PI3K and interactive pathways that might
lead to targeted therapy opportunities in clinical practice.

DESIGN, SETTING, AND PARTICIPANTS From January 2013 through December 2014, 19 784
consecutive tumor samples (>40 cancer types) were sent from thousands of clinicians in 60
countries to a single commercial laboratory for molecular profiling, including next generation
sequencing, protein expression (immunohistochemical analysis [IHC]), and gene
amplification (fluorescent in situ hybridization or chromogenic in situ hybridization).

MAIN OUTCOMES AND MEASURES Patterns in targetable genomic and proteomic alterations in
the PI3K pathway and coincidence with hormone receptor and HER2 alterations.

EXPOSURES Molecular profiling across solid tumors.

RESULTS Overall, 38% of patients had an alteration in 1 or more PI3K pathway components,
most commonly phosphatase and tensin homologue (PTEN) loss (by IHC) (30% of all
patients), followed by mutations in PIK3CA (13%), PTEN (6%), or AKT1 (1%). Seventy percent
of patients with endometrial cancer and more than 50% of patients with breast, prostate,
anal, hepatocellular, colorectal, and cervical cancer exhibited alterations in at least 1 PI3K
pathway gene and/or gene product. Examples of frequent aberrations included PTEN loss in
hepatocellular (57% of patients), colorectal (48%), gastric (36%), prostate (52%), and
endometrial cancer (49%); PIK3CA mutations in endometrial (37%), breast (31%), cervical
(29%), and anal cancer (27%). PIK3CA, PTEN, and AKT1 mutations occurred more frequently
in the presence of hormone receptor overexpression (androgen, progesterone, or estrogen
receptor). PIK3CA mutations were also more common in the HER2-positive than in the
HER2-negative group; the opposite pattern was seen for PTEN mutation or PTEN loss.

CONCLUSIONS AND RELEVANCE PI3K pathway aberrations are among the most common in
cancer. They do not segregate by classic cancer histologic characteristics. Patterns of Author Affiliations: Caris Life
biomarker coalterations involving HER2 and hormone receptors may be important for Sciences, Phoenix, Arizona (Millis,
Reddy, Gatalica); Cancer Center,
optimizing combination treatments across cancer types. Tokyo Medical and Dental University,
Tokyo, Japan (Ikeda); Division of
Hematology/Oncology, Department
of Medicine, University of
California–San Diego, La Jolla (Ikeda,
Kurzrock); Center for Personalized
Cancer Therapy, University of
California–San Diego, La Jolla (Ikeda,
Kurzrock); Moores Cancer Center,
University of California–San Diego,
La Jolla (Ikeda, Kurzrock).
Corresponding Author: Sadakatsu
Ikeda, MD, Moores Cancer Center,
University of California–San Diego,
3855 Health Sciences Dr, Rm 2306,
JAMA Oncol. 2016;2(12):1565-1573. doi:10.1001/jamaoncol.2016.0891 La Jolla, CA 92093-0658 (saikeda
Published online July 7, 2016. @ucsd.edu).

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 Research Original Investigation
T
he phosphatidylinositol 3-kinase (PI3K) pathway is one
of the most commonly activated signals in diverse can-
cer types.1 This pathway controls cell proliferation,
growth, differentiation, protein synthesis, glucose metabo-
lism, migration, and apoptosis.1 Activation of this pathway is
initiated by the binding of corresponding ligands to tyrosine
kinase receptors.1 A regulatory subunit of PI3K is phosphory-
lated and results in the activation of p110, a catalytic subunit
of PI3K.2 The activation of PI3K leads to production of phos-
phatidylinositol 3,4,5-triphosphate, a lipid second messen-
ger, and further activation of downstream effectors such as pro-
tein kinase B (AKT) and mammalian target of rapamycin
(mTOR).3 Phosphatase and tensin homologue (PTEN) dephos-
phorylates proteins in this pathway.3
The PI3K pathway can be constitutively activated by ge-
nomic aberrations in cancer.4 Common alterations include (1)
activating mutations or/and amplification of the catalytic sub-
unit alpha (PIK3CA),5,6 (2) loss of PTEN,7 and (3) mutation
and/or amplification of AKT, a serine/threonine-specific pro-
tein kinase.8 These alterations are sufficient to induce tumori-
genesis in preclinical models.9-11 Genomic alterations of the
PI3K pathway have been observed in diverse solid tumors, in-
cluding, but not limited to, breast, endometrial, epithelial ovar-
ian, prostate, bladder, colorectal, gastric, and pancreatic can-
cer, as well as various squamous cell cancers, melanoma, and
glioblastoma.12-14 However, these studies generally had small
sample sizes, and it is difficult to capture the true frequency
of alterations owing to the different techniques used in each
study. The Cancer Genome Atlas has provided the most com-
prehensive evaluation to date, including an analysis of 12 ma-
jor cancers and several studies in individual cancers.15-17
Importantly, the PI3K/AKT/mTOR machinery often does
not act alone. For instance, PIK3CA mutations induce resis-
tance to anti-HER2 (human epidermal growth factor receptor
2) treatments.18 PIK3CA mutations have also been correlated
with hormone-receptor positivity in breast cancer, and the
combination of the mTOR inhibitor everolimus with hor-
mone modulators have shown clinical efficacy in breast and
other cancers, though responses were not clearly associated
with mutation status in some studies.19-21
In the present study, we profile a large number of diverse
solid tumors in a single laboratory certified by the Clinical Labo-
ratory Improvement Amendment (CLIA). We catalogue the ge-
nomic abnormalities in several key members of the PI3K path-
way as well as coexisting anomalies, including those in
hormone and HER2 receptors.
Methods
Tissue Samples
Consecutive cases submitted to a commercial CLIA-certified
laboratory (Caris Life Sciences) from January 2013 through De-
cember 2014 were analyzed. The tissue diagnoses were sub-
mitted based on pathologic assessment of physicians who re-
quested the assays and were further verified by a pathologist
at the Caris laboratory. Formalin-fixed paraffin-embedded tis-
sues were processed as previously described.22 In accor-
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PI3K Pathway Alterations in Diverse Tumors
Key Points
Question What are the frequencies of aberrations in the
phosphatidylinositol-3-kinase (PI3K)/protein kinase B
(AKT)/mammalian target of rapamycin (mTOR) pathway across
diverse solid tumors?
Findings In this retrospective analysis of 19 784 patients,
aberrations in the PI3K/AKT/mTOR pathway were identified in
38%, across solid tumor histologic findings. These alterations also
exhibit patterns of coalterations with ERBB2/HER2 (formerly HER2
or HER2/neu) and hormone receptors; human epidermal growth
factor receptor 2 (HER2)- and hormone-positive tumors exhibit
higher rates of PI3K/AKT/mTOR alterations.
Meaning The patterns may be important for clinical trial
development and for optimizing combination treatments across
cancer types.
dance with Western Institutional Review Board guidelines,
patient identity protection was maintained throughout the
study, so the study was considered exempt, and institutional
review board approval was waived. Samples were sent from
thousands of clinicians and institutions in 60 countries.
Next-Generation Sequencing
Next generation sequencing (NGS) analysis was performed
on genomic DNA isolated from formalin-fixed paraffin-
embedded tissue using the MiSeq platform (Illumina). An Agi-
lent custom-designed SureSelect XT assay was used to enrich
a targeted NGS hotspot panel (47 genes). All variants reported
by this assay were detected with greater than 99% confi-
dence, based on the frequency of the mutation present and the
amplicon coverage. The average depth of coverage for this as-
say is 1000×. Using the COSMIC database (Catalogue of
Somatic Mutations in Cancer; http://cancer.sanger.ac.uk
/cosmic), we did not report mutations if the alteration was con-
sidered benign. Known pathogenic variants, presumed patho-
genic variants, and variants of unknown significance were
included in the analysis. This test was not designed to distin-
guish between germline inheritance of a variant or acquired
somatic mutation; it has the sensitivity to detect approxi-
mately a 10% population of cells containing a mutation.
Immunohistochemical Analysis
Immunohistochemical analysis (IHC) was performed on the
tumor samples using commercially available detection kits and
autostainers (Benchmark XT, Ventana Medical Systems Inc, and
Autostainer Link 48, Dako). Primary antibodies used for pro-
tein detection were human epidermal growth factor receptor
2 (HER2) (clone 4B5, Ventana), PTEN (clone 6H2.1, Dako), es-
trogen receptor (ER) (clone SP1, Ventana), progesterone recep-
tor (PR) (clone 1E2, Ventana), and androgen receptor (AR) (clone
AR27, Leica Biosystems). The thresholds used to define posi-
tivity are described in eTable 1 in the Supplement. Loss of PTEN
was defined as no protein expression in more than 50% of cells
by IHC. Expression of PTEN was assessed based on the stain-
ing of cytoplasm and/or nucleus of the neoplastic cells. Ab-
sence of staining in any of the subcellular compartments was
recorded as 0; when present, the intensity of staining in either
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 PI3K Pathway Alterations in Diverse Tumors Original Investigation Research

Table 1. Frequency of Alterations in PIK3CA, PTEN, and AKT1 Across Diverse Solid Cancersa

Cancer Specimens, %
Total Cases, PIK3CA PTEN PTEN AKT1
Cancer Type No. Mutated Loss Mutated Mutated
Total 19 784 13 30 6 1
Endometrial carcinoma 1616 37 49 33 3
Breast cancer 2333 31 34 5 3
Cervical cancer 291 29 30 4 1
Anal cancer 71 27 31 3 1
Bladder cancer 313 22 31 4 1
Colorectal cancer 1991 17 48 3 1
Head and neck squamous cell carcinoma 234 14 32 4 2
Nonmelanoma skin cancer 92 10 26 0 1
Salivary gland cancer 66 10 22 2 0
Unknown primary 638 10 27 3 2
Glioblastoma 529 9 6 13 0
Rare, others 126 8 22 2 1
Epithelial ovarian cancer 3539 8 21 3 1
Gastric cancer 221 7 36 2 0
Gallbladder cancer 72 7 35 3 0
Prostate cancer 173 6 52 14 1
Nonepithelial ovarian cancer 141 6 28 1 2
Cholangiocarcinoma 169 6 30 2 1
Lung, non–small-cell lung cancer 2391 5 31 2 1
Kidney cancer, clear cell RCC 153 5 44 3 0
Esophageal, GE junction cancer 300 5 35 2 0
Appendiceal cancer 188 4 28 3 1
Soft-tissue sarcoma 679 4 14 2 0
Uterine sarcoma 337 4 11 4 1
Liver, hepatocellular carcinoma 116 4 57 2 1
Adenoid cystic carcinoma 67 3 9 0 4
Neuroendocrine tumor 381 3 7 2 1
Gastrointestinal stromal tumor 71 3 2 0 0
Pancreatic cancer 761 3 34 1 0
Bone cancer 72 3 19 3 0 Abbreviations: PTEN, phosphatase
and tensin homologue; RCC, renal cell
Melanoma 668 2 24 5 0
carcinoma.
Lung, small-cell lung cancer 157 2 24 5 0 a
Results from cancers with more
Low-grade glioma 73 1 6 4 0 than 50 samples are shown here.
Thyroid cancer 82 1 12 5 2 Categories with fewer than 50 cases
Mesothelioma 114 0 13 1 0 are listed in eTable 2 in the
Supplement.

cytoplasm or nucleus was graded from 1+ to 3+, based on the
comparison with the internal positive normal cells, which for
the purpose of the study were endothelial cells and periph-
eral nerves present in the section. The percentage of cancer
cells with any level positivity was recorded. The findings of
PTEN IHC were validated in a cohort of samples containing 43
cases (23 negatives and 21 positives) stained at our institution
and compared with the staining results performed at another
reference CLIA-accredited laboratory.
ratio higher than 2.2 was considered amplified. CISH was
performed using the INFORM HER2 Dual ISH DNA Probe Cock-
tail (Ventana) according to the manufacture’s protocol. A HER2/
CEP17 ratio higher than 2.0 was considered amplified.
Statistical Analysis
A Fisher exact test with a 2-tailed P value was used to com-
pare biomarker differences across histologic subtypes, using
GraphPad software, version 6.00 (August 2012).

In Situ Hybridization
Either fluorescence in situ hybridization (FISH) or chromo-
genic in situ hybridization (CISH) was performed to detect copy
number changes in ERBB2/HER2 (formerly HER2 or HER2/
neu). FISH was performed using the Pathvysion HER2 DNA
Probe Kit (Abbott Laboratories). Interphase nuclei were ex-
amined. The signal of ERBB2/HER2 was compared with chro-
mosome 17 centromere signals (CEP17), and a HER2/CEP17
Results
Alterations in the PI3K pathway were common across cancers.
Indeed, of 19 784 tumors analyzed, 13% had PIK3CA muta-
tions; 30%, PTEN loss; 6%, PTEN mutations; and 1%, AKT1
mutations (Table 1). Thirty-eight percent of patients had an
aberration in 1 or more of these genes and/or gene products

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 Research Original Investigation
(Figure 1A). In endometrial cancer, 70% of tumors had an al-
teration in at least 1 of these pathway genes and/or gene prod
ucts, and in breast, prostate, anal, hepatocellular, colorectal, and
cervical cancer, over 50% exhibited 1 or more aberrations.
PIK3CA Mutations
PIK3CA mutations were observed in 0% to 37% of solid
tumors (Figure 1, Table 1; eTable 2 in the Supplement). The most
commonly found mutated cancers were endometrial (37% of
patients), breast (31%), cervical (29%), anal (27%), and blad-
der cancer (22%). Other cancers with identified mutations in
more than 10% of cases included colorectal (17%), sarcoma-
toid renal cell carcinoma (15%), head and neck squamous cell
carcinoma (14%), cancers of unknown primary (10%), sali-
vary gland (10%), and nonmelanoma skin cancer (10%).
PTEN Loss and PTEN Mutations
The loss of PTEN was common, ranging from 57% in hepato-
cellular carcinoma to 2% in gastrointestinal stromal tumor
(Figure 1, Table 1; eTable 2 in the Supplement). Frequent loss
was also observed in ampullary adenocarcinoma (53%), pros-
tate cancer (52%), germ cell tumors (50%), endometrial car-
cinoma (49%), colorectal cancer (48%), small intestinal can-
cer (40%), clear cell renal cell carcinoma (44%), extrahepatic
cholangiocarcinoma (37%), esophageal and/or gastroesopha-
geal junction cancer (35%), gastric cancer (36%), pancreatic
cancer (32%), breast cancer (34%), and bladder cancer (31%).
Loss of PTEN was less frequent in thymic carcinoma (9%), neu-
roendocrine tumors (7%), central nervous system tumors
(6%), uveal melanoma (4%), and gastrointestinal stromal tu-
mor (2%). PTEN mutation was less frequent than PTEN loss,
affecting 0% to 14% of patients, except for endometrial can-
cer, in which 33% of individuals had PTEN mutations.
AKT1 Mutations
AKT1 mutations were infrequent, ranging from 0% to 4% of
cases. Adenoid cystic carcinoma and thyroid carcinoma (4%
of patients) were the most commonly mutated cancers, fol-
lowed by adrenocortical (3%), breast (3%), and endometrial
cancer (3%). Amplification of AKT1 was not evaluated.
PI3K/AKT/mTOR Combined Genomic Alterations
Aberrations of the PI3K/AKT/mTOR pathway were observed
in 38% of solid tumors. As noted with PIK3CA mutations
alone, epithelial cancers such as endometrial cancer, breast
cancer, and gastrointestinal tract cancer were most com-
monly associated with genomic alterations in all the evalu-
ated PI3K pathway biomarkers. Nonepithelial tumors such as
melanoma, sarcoma, and neuroendocrine tumors harbored
PI3K pathway alterations less often.
Mutation Hot Spots
PIK3CA mutations were observed most frequently in exon 9
(43.2%) and exon 20 (33.0%) (eFigure 1 in the Supplement).
Specifically, E545K substitution in exon 9 accounted for 20.5%,
and H1047R in exon 20 accounted for 20.6% of mutations in
analyzed samples. Position 545 is within the helical domain
and interacts with the N-terminal SH2 domain of the p85 regu-
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PI3K Pathway Alterations in Diverse Tumors
latory subunit.23 The change from glutamic acid (E) to lysine
(K) causes charge reversal and disrupts the inhibitory inter-
action with p85.5 The mutation of E545K was sufficient to in-
duce tumorigenesis in a transgenic mouse model.24,25
Position 1047 resides in the kinase domain and is located
near the C-terminal end of the activation loop.23 Substitution
of histidine (H) to arginine (R) in this position likely changes the
conformation of the activation loop 23 and results in the
activation of the PI3K pathway.25 The mutation of H1047R was
sufficient to induce tumors in an animal model26 and may be
associated with favorable responses to PI3K/AKT/mTOR
pathway inhibitors.27
The most common PTEN mutation hot spot was a K267
frameshift mutation (6.6%) in exon 7. Position K267 resides in
the calcium-binding region 3 (CRB3) loop of the C2 domain.28
The mutation of K267 is sufficient to block PTEN membrane
localization.28
Coexistence of PTEN Mutation, PTEN Loss, PIK3CA
Mutation, and AKT1 Mutation
Loss of PTEN protein expression was the most common aber-
ration (30% of patients), followed by PIK3CA mutation, and
PTEN mutation (Table 1, Figure 1 and Figure 2). AKT1 muta-
tion is a relatively rare event (only 1% of patients). PTEN mu-
tation frequently occurs with PTEN loss.
Loss of PTEN expression is, however, not always associ-
ated with PTEN mutation. Only 13% of patients with PTEN loss
had a PTEN mutation (675 of 5005); on the other hand, 72.5%
of patients with PTEN mutation had PTEN loss (675 of 931)
(Figure 2). Fourteen percent of patients (700 of 5005) with
PTEN loss also had a PIK3CA mutation; 32% of patients (296
of 931) with a PTEN mutation also had a PIK3CA mutation; and
14% of patients (296 of 2156) with PIK3CA mutations also had
PTEN mutations.
Association With Hormone-Receptor Pathways
It is well recognized that the PI3K/AKT/mTOR pathway inter-
acts with other signaling pathways. Our IHC studies of key sig-
naling pathways revealed increased expression of the AR, ER,
and PR in PIK3CA-mutated samples compared with PIK3CA
wild-type samples (Table 2). Androgen receptor was overex-
pressed in 29% of PIK3CA-mutated cases, whereas overex-
pression of AR was seen in only 16% of PIK3CA wild-type
samples (P < .001). Similarly, ER and PR were expressed more
commonly in PIK3CA-mutated cases than in PIK3CA wild-
type cases (ER, 44% of PIK3CA-mutant cases overexpressed
ER, whereas only 23% of PIK3CA wild-type cases overex-
pressed ER [P < .001]; PR, 33% vs 13% [P < .001]) (Table 3). Con-
versely, in ER-positive patients, PIK3CA was mutated in 23%
of cases, while in ER-negative patients, PIK3CA was mutated
in only 11% of cases (P < .001) (Figure 3A). These results indi-
cate a strong interaction between the PI3K/AKT/mTOR path-
way and the hormone-receptor pathways.
Coalterations With HER2
In this study, patients with overexpressed or amplified
HER2 also more frequently had PIK3CA mutations than did
HER2-normal patients (22% vs 13%; P < .001) (Figure 3B).
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 PI3K Pathway Alterations in Diverse Tumors Original Investigation Research

Figure 1. Frequency of Genomic Alterations in the Phosphatidylinositol-3-Kinase (PI3K) Pathway

A PI3K pathway aberrations

All, composite (N = 19 784)

Endometrial carcinoma (n = 1616)
Breast cancer (n = 2333)
Prostate cancer (n = 173)
Anal cancer (n = 71)
Liver, hepatocellular carcinoma (n = 116)
Colorectal cancer (n = 1991)
Cervical cancer (n = 291)
Kidney cancer, ccRCC (n = 153)
Bladder cancer (n = 313)
Head and neck squamous cell carcinoma (n = 234)
Gallbladder cancer (n = 72)
Gastric cancer (n = 221)
Esophageal, GE junction cancer (n = 300)
Nonepithelial ovarian cancer (n = 141)
Pancreatic adenocarcinoma (n = 761)
Unknown primary (n = 638)
Nonmelanoma skin cancers (n = 92)
Cholangiocarcinoma (n = 169)
Appendiceal cancer (n = 188)
Vulvar cancer (n = 41)
Lung, NSCLC (n = 2391)
Salivary gland cancer (n = 66)
Rare, others (n = 126)
Ovarian epithelial carcinoma (n = 3539)
Lung, SCLC (n = 157)
Glioblastoma (n = 529)
Melanoma (n = 668)
Bone cancer (n = 72)
Soft-tissue sarcoma (n = 679)
Uterine sarcoma (n = 337)
Thyroid cancer (n = 82)
Adenoid cystic carcinoma (n = 67)
Mesothelioma (n = 114)
Neuroendocrine tumor (n = 381)
Low-grade glioma (n = 73)
Gastrointestinal stromal tumor (n = 71)

0 10 20 30 40 50 60 70 80
Frequency, %

B PIK3CA and PTEN mutations

All, composite (N = 19 784)
Endometrial carcinoma (n = 1616)
Breast cancer (n = 2333)
Cervical cancer (n = 291)
Anal cancer (n = 71)
Bladder cancer (n = 313)
Colorectal cancer (n = 1991)
Head and neck squamous cell carcinoma (n = 234)
Nonmelanoma skin cancers (n = 92)
Salivary gland cancer (n = 66)
Unknown primary (n = 638)
Glioblastoma (n = 529)
Rare, others (n = 126)
Ovarian epithelial carcinoma (n = 3539)
The numbers of patients tested in
Gastric cancer (n = 221)
Gallbladder cancer (n = 72) each lineage (solid tumors with more
Prostate cancer (n = 173) than 50 cases tested) are identified in
Nonepithelial ovarian cancer (n = 141) parentheses. Categories with fewer
Uveal melanoma (n = 36) than 50 cases are detailed in eTable 2
Cholangiocarcinoma (n = 169)
in the Supplement. A, Frequency of
Lung, NSCLC (n = 2391)
Kidney cancer, ccRCC (n = 153) PI3K pathway aberrations in solid
Esophageal, GE junction cancer (n = 300) tumors; the percentages represent
Appendiceal cancer (n = 188) the combined total phosphatase and
Soft-tissue sarcoma (n = 679) tensin homologue (PTEN) loss, as
Uterine sarcomas (n = 337)
well as PTEN, PIK3CA, and AKT1
Liver, hepatocellular carcinoma (n = 116)
Adenoid cystic carcinoma (n = 67) mutations found in patients. B,
Neuroendocrine tumor (n = 381) Frequency of PIK3CA and PTEN
Gastrointestinal stromal tumor (n = 71) mutations in various solid tumors,
Pancreatic adenocarcinoma (n = 761) listed by decreasing frequency of
Bone cancer (n = 72) Mutated PTEN PIK3CA mutation. Numbers in
Melanoma (n = 668)
Lung, SCLC (n = 157) Mutated PIK3CA parenthesis are the numbers of
Low-grade glioma (n = 73) patients tested. ccRCC indicates
Thyroid carcinoma (n = 82) clear-cell renal-cell carcinoma; GE,
Mesothelioma (n = 114) gastroesophageal; GIST,
0 5 10 15 20 25 30 35 40 gastrointestinal stromal tumor;
Frequency, % NSCLC, non–small-cell lung cancer;
SCLC, small-cell lung cancer.

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 Research Original Investigation PI3K Pathway Alterations in Diverse Tumors

HER2 overexpression or amplification is associated with de-
creased frequency of PTEN loss (27% vs 30% when HER2 sta-
tus is normal, P < .001. Figure 3B). Additional analysis of the
HER2-positive PIK3CA- or PTEN-mutated cases was per-
formed to assess differences in expression of ER and identi-
fied that: of 2237 patients with a PIK3CA mutation, 193 (8.6%)
were also ER positive and HER2 positive, and 183 (8.1%) were
ER negative and HER2 positive; of the 916 PTEN-mutant pa-
tients, 36 (3.9%) were ER positive and HER2 positive, and 25
(2.7%) were ER negative and HER2 positive.

Figure 2. Coexistence of PTEN and PIK3CA Mutations and PTEN Loss

Discussion
PTEN mutant
(n = 931) To our knowledge, this is the largest study ever to provide the
189 frequency of genomic and proteomic alterations in a variety of
67 PI3K/AKT/mTOR-relevant pathways. The analysis was per-
PTEN loss 446
(n = 5005) 229 formed at a CLIA-certified laboratory with consistency in tech-
1389 niques and platforms. The large number of samples is likely
3859 471
sufficient to capture the true frequency of these alterations,
and provide a reference guide for deployment of targeted
PIK3CA mutant therapies.
(n = 2156) Conclusions from many previous studies have been lim-
ited owing to small sample sizes and the different methods used
to measure alterations. For example, the published fre-
Venn diagram showcases the co-incidence of PTEN mutations, PIK3CA mutations,
and PTEN loss. The number represents sample numbers, in which all 3 biomarkers
quency of PTEN loss in prostate cancer varies from 13% to
were evaluated from a total of 17 546 cases. For example, of 5005 cases with 65%.29-34 These discrepant frequencies could be explained by
phosphatase and tensin homologue (PTEN) loss, 3859 had neither PTEN several factors: (1) small number of specimens (range, 22-
mutations nor PIK3CA mutations, while 675 cases had a PTEN mutation (446 with
80); (2) differences in disease stage of the specimens; and (3)
only a PTEN mutation, and 229 with both a PTEN mutation and a PIK3CA
mutation). Therefore, only 13% of patients with PTEN loss had a PTEN mutation the distinct techniques that were used in these studies. In ad-
(675 of 5005); on the other hand, 72.5% of patients with PTEN mutation had dition, cutoff points for designating PTEN loss may vary. We
PTEN loss (675 of 931). Another 471 cases with PTEN loss had only a PIK3CA defined PTEN loss as no protein expression in more than 50%
mutation. (Owing to low incidence, AKT1 is not shown in the Venn diagram.)
of cells by IHC. Some studies may have a more strict defini-

Table 2. Frequency of Gene Mutations by PIK3CA and PTEN Statusa

ERBB2/
Gene TP53 BRAF KRAS HRAS FBXW7 FGFR2 HNF1A ATM CTNNB1 HER2 PTEN PIK3CA
PIK3CA
Wild type 49 4 16 1 2 1 1 3 2 1 5 NA
Mutated 35 2 21 1 6 3 2b 4 7 2 14 NA
PTEN
Wild type 47 4 17 1 2 1 1 3 2 1 NA 12
Mutated 34 4b 19b 0.4b 6 7 5 5 12 2 NA 32
a
All evaluations performed by next-generation sequencing mutation analysis, and data reported as frequency percentages.
b
Differences between wild-type and mutated PIK3CA and PTEN genes was not significant; in all other cases they were (P<.001).

Table 3. Protein Expression Levels or Copy Number Increase by PIK3CA and PTEN Status

IHC Protein Expression Above Threshold, %a ISH Amplification, %

ERBB2/
Gene PTEN Loss TOP2A AR ER PR MGMT PGP TS HER2 HER2 cMET
PIK3CA
Wild type 29 73 16 23 13 58 17 49 6 4 1
Mutated 31 86 29 44 33 54 12 52 11 6 0.2
PTEN
Wild type 25 73 18 24 14 58 17 49 7 4 1
Mutated 72.5 84 23 49 42 42 10 60 4 1 0.2
a
Abbreviations: AR, androgen receptor; cMET, MET proto-oncogene, receptor Thresholds for positivity are detailed in eTable 1 in the Supplement; For PTEN,
tyrosine kinase; ER, estrogen receptor; HER2, human epidermal growth factor MGMT, PGP, and TS, loss of expression or expression levels below threshold
receptor 2; IHC, immunohistochemical analysis; ISH, in situ hybridization; are indicative of response to therapy. For this table, as an example, TP53 is
MGMT, O(6)-methylguanine-DNA methyltransferase; NA, not applicable; mutated in 49% of patients with wild-type PIK3CA and in 35% with PIK3CA
NGS, next-generation sequencing; PGP, P-glycoprotein 1; PR, progesterone mutations.
receptor; PTEN, phosphatase and tensin homologue; TOP2A, DNA
topoisomerase 2-alpha; TS, thymidylate synthase.

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 PI3K Pathway Alterations in Diverse Tumors
Figure 3. Association of PIK3CA, PTEN, and AKT1 Mutations and PTEN Loss by Hormone Receptor (A) and HER2 Status (B)
A Hormone receptor status
35
Overall
30 AR+
AR−
25
ER+
Frequency, %
ER−
20
PR+
15 PR−
10
5
0 0
AKT1 mut PIK3CA mut PTEN mut
Bars represent frequency of genetic aberrations. AR indicates androgen receptor; ER, estrogen receptor; HER2, human epidermal growth factor receptor 2;
IHC, immunohistochemical analysis; ISH, in situ hybridization; mut, mutated; PR, progesterone receptor; PTEN, phosphatase and tensin homologue; plus sign,
positive status; minus sign, negative status.
tion, and therefore the incidence of PTEN loss may be lower
in those studies.
Specific to molecular alterations, large-scale data are
available from The Cancer Genome Atlas (TCGA) (http:
//cancergenome.nih.gov/), and an analysis using these data was
performed by Kandoth et al.16 Overall, PIK3CA was mutated
in 13% of our patients vs about 18% of the TCGA patients; PTEN
was mutated in about 6% in the present study vs approximately
10% in the TCGA; and in both data sets, AKT mutation rates
were about 1% (eTable 3 in the Supplement).16 Differences in
overall rates of mutations could be due to several factors
including, but not limited to, the large numbers of diverse
cancers (including rare tumors) assessed in our data set (>40
types of cancer vs the 12 cancer types assessed by Kandoth and
colleagues), and the difference in patient numbers (19 784 vs
3281 patients).16 Loss of PTEN identified by IHC was seen in
about 30% of our patients and was not assessed in TCGA.
Compared with the TCGA pan-cancer analysis, our study
demonstrated similar mutation rates in cancers such as breast,
colorectal, bladder, lung, and renal cell carcinoma.
Recently, the PI3K pathway has been conjectured to be im-
portant in the clinic, and agents targeting the PI3K/AKT/
mTOR machinery have entered the clinical arena.35 In breast
cancer, treatment with everolimus, an mTOR inhibitor, and ex-
emestane (hormone modulator) demonstrated prolonged over-
all survival in hormone receptor–positive metastatic breast can-
cer and was approved by the US Food and Drug Administration
(FDA).20 Everolimus is also approved by the FDA for renal cell
carcinoma, pancreatic neuroendocrine tumor, and subepen-
dymal giant cell astrocytoma.36-38 In hematologic cancers, ide-
lalisib, a PI3K delta inhibitor, is approved by the FDA for chronic
lymphocytic leukemia, small lymphocytic lymphoma, and fol-
licular lymphoma.39 Interestingly, aspirin use in patients with
colorectal cancer who harbor PIK3CA mutations was associ-
ated with superior survival in an observational study, per-
haps by inhibiting of cyclooxygenase-2, which is potentiated
by PI3K mutations.40 Targeting the PI3K pathway is currently
jamaoncology.com
Copyright 2016 American Medical Association. All rights reserved.
Downloaded From: on 08/22/2018
Original Investigation Research
B HER2 status
30
Overall HER2− HER2+
(IHC or ISH) (IHC or ISH)
25
20
Frequency, %
15
10
5
PTEN loss PIK3CA mut PTEN mut AKT1 mut PTEN loss
being investigated in multiple tumor types. The list, while not
exhaustive, includes prostate cancer, 41,42 endometrial
cancer,21,43 non–small-cell lung cancer,44,45 colon cancer,46,47
gastric cancer,48,49 cervical cancer,50 hepatocellular carcinoma,51
lymphoid cancers,52,53 and PI3K pathway–altered cancers.54 Of
interest in this regard are also virally associated cancers. Can-
cers associated with human papillomavirus often show PIK3CA
pathway mutations in, for instance, head and neck cancer, but
patients with this type of tumor that is not virally mediated have
a distinct genomic portfolio.15,55
In addition to histology oriented trials, biomarker-driven
trials that target the PI3K pathway-associated gene aberra-
tions are under way.56 A cautionary note, however, should be
mentioned. The initial clinical trials demonstrate that single-
agent inhibition of the PI3K/AKT/mTOR pathway is unlikely
to be effective in the vast majority of advanced cancers,35 per-
haps because of the cooccurrence of RAS/RAF alterations57 or
anomalies in the hormone receptor pathways and/or in HER2,
as observed in this analysis and by others.58-61 Indeed, the re-
cent SHIVA trial62,63 showed disappointing results with the use
of single agents in advanced cancers, including everolimus for
patients with PI3K/AKT/mTOR anomalies. These observa-
tions suggest that optimal deployment of combination regi-
mens may need to take into consideration the crosstalk
between aberrant signals, in addition to consideration of the
specific alteration, which may affect efficacy, as well.
Our analysis showed that PTEN loss occurred commonly
in the absence of PTEN mutations. Indeed, only 13% of speci-
mens with PTEN loss harbored PTEN mutations (hotspot mu-
tation analysis, not entire gene). Other mechanisms for PTEN
loss are known, including epigenetic silencing,64 posttranscrip-
tional modification by microRNA,65,66 and posttranslational
modification by proteosomal degradation.67 Of interest, PTEN
inactivation is sometimes reported to be mutually exclusive
with PIK3CA mutations, but we found a 4% cooccurrence
(patients with PTEN loss or PTEN mutation plus PIK3CA
mutation divided by the total tested for PTEN/PIK3CA/
(Reprinted) JAMA Oncology December 2016 Volume 2, Number 12 1571
 Research Original Investigation PI3K Pathway Alterations in Diverse Tumors

AKT mutation (n = 17 546) (Figure 2). This raises the possibil-
ity that some of the mutations do not affect PI3K signaling. Al-
though our analysis did not examine function, a more detailed
assessment of this possibility merits future investigation.
Crosstalk between the hormone receptor pathways and the
PI3K/AKT/mTOR pathway, as shown in this study, supports pre-
vious work.19 In this study, mutations of PIK3CA and PTEN
were more frequently discerned in patients with higher ex-
pression of ER, PR, and AR. Interestingly, PTEN loss was in-
versely correlated with ER and AR expression (though PTEN
loss was still found in both ER-positive and ER-negative tu-
mors as well as AR-positive and AR-negative cancers; Figure 3A
and Table 2).
In general, KRAS and EGFR mutations are mutually
exclusive.68 In our analysis, mutation of PIK3CA was associ-
ated with higher frequency of KRAS mutations. Also, muta-
tions of PIK3CA were associated with increased levels of HER2
and other markers such as DNA topoisomerase 2-alpha (TOP2A)
(Tables 2 and 3). TOP2A possibly predicts vulnerability to an-
thracyclines and etoposide, setting the stage for rational com-
binations. These results imply that alterations of the PI3K ma-
chinery are not exclusive of other key signaling pathways but
rather are associated with a high chance of crosstalk with other
pathways. A similar observation was reported in a study of lung
cancer.64 When making treatment decisions based on molecu-
lar profile, multiple alterations should be considered prior to
determining therapy.
Conclusions
In conclusion, this large, single laboratory study helps so-
lidify the frequency of PI3K pathway aberrations across
tumor types and the coexisting patterns of alterations in a va-
riety of other markers, including hormone receptors and HER2.
The data presented here can be used to help design clinical
studies that appropriately investigate and treat tumors with
targeted agents.

ARTICLE INFORMATION REFERENCES 12. Wu G, Xing M, Mambo E, et al. Somatic

Accepted for Publication: March 7, 2016.
Published Online: July 7, 2016.
doi:10.1001/jamaoncol.2016.0891.
Open Access: This article is published under JAMA
Oncology’s open access model and is free to read on
the day of publication.
Author Contributions: Dr Millis had full access to
all of the data in the study and takes responsibility
for the integrity of the data and the accuracy of the
data analysis. Drs Millis and Ikeda contributed
equally to this work.
Study concept and design: Millis, Ikeda, Reddy,
Kurzrock.
Acquisition, analysis, or interpretation of data: Millis,
Ikeda, Reddy, Gatalica, Kurzrock.
Drafting of the manuscript: Millis, Ikeda.
Critical revision of the manuscript for important
intellectual content: Millis, Ikeda, Reddy, Gatalica,
Kurzrock.
Statistical analysis: Millis.
Administrative, technical, or material support: Millis,
Ikeda, Reddy.
Study supervision: Millis, Reddy, Kurzrock.
Conflict of Interest Disclosures: Dr Millis was
previously employed by Caris Life Sciences, a
for-profit company. Drs Reddy and Gatalica are
employed by Caris Life Sciences. Dr Kurzrock has
research funding from Genentech, Merck Serono,
Pfizer, Sequenom, Foundation Medicine, and
Guardant, as well as consultant fees from
Sequenom and an ownership interest in Novena Inc
and CureMatch Inc. No other disclosures are
reported.
Funding/Support: This study was funded in part by
the Joan and Irwin Jacobs Fund.
Role of the Funder/Sponsor: The Joan and Irwin
Jacobs Fund had no role in design and conduct of
the study; collection, management, analysis, and
interpretation of the data; preparation, review, or
approval of the manuscript; and decision to submit
the manuscript for publication.
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