Supplementary Methods
Definition of dose-limiting toxicities
Dose-limiting toxicities (DLTs) were defined as any of the following occurring in Cycle 1: an
adverse event (AE) that warranted a dose reduction (determined by the investigator); an AE
that was of potential clinical significance such that further dose escalation would expose
patients to unacceptable risk (determined by the cohort review committee); grade 3/4 nonhematologic AE occurring despite optimal prophylaxis or not easily managed or corrected by
medical intervention; grade 4 neutropenia for ≥4 days, grade 3/4 febrile neutropenia for ≥3
days; any other hematologic toxicity of grade 4; recurrent grade 2 or 3 hyperglycemia
despite appropriate treatment with an oral hypoglycemic agent (e.g. metformin); or inability
to take ≥75% of planned study drug doses in the first 28 days of study treatment because of
an AE.
Pharmacodynamic assessments
Blood was collected for fasting plasma glucose analysis in 2 mL BD Vacutainer tubes with
glycolytic inhibitor, 3.0 mg sodium fluoride, 6.0 mg Na2EDTA (Becton-Dickinson
cat#367587). Following centrifugation at room temperature in a horizontal rotor (swing-out
head) for 15 minutes at 1500 to 2000 relative centrifuge force, plasma (top layer) was
transferred into a 5 mL plastic vial. Plasma samples were analyzed at Covance Central
Laboratory (GLUC3 [Glucose HK Gen. 3] reagent kit used with Roche Modular and Cobas
Analyzer).
Blood was collected for C-peptide analysis in 6 mL BD Vacutainer tubes with K2EDTA
(Becton-Dickinson cat#367863). Following centrifugation at room temperature in a horizontal
rotor (swing-out head) for 15 minutes at 1200 relative centrifuge force, plasma (top layer)
was collected, snap-frozen in liquid nitrogen or on dry ice, and stored as frozen aliquots at
−70°C (or below). Plasma samples were analyzed at MLM Medical Labs GmbH (Electro
Chemiluminescence Immuno Assay [ECLIA] assay from Roche Diagnostics).
Molecular profiling of formalin-fixed paraffin-embedded tumor sections
Phosphatase and tensin homolog immunohistochemical analysis
Phosphatase and tensin homolog (PTEN) expression was evaluated at Sanofi in formalinfixed paraffin-embedded (FFPE) tissue sections (5 micron) with an anti-PTEN antibody.
Hematoxylin and eosin (H&E) stains were performed on tissue sections to examine
specimen quality and appropriateness. The PTEN analyses were carried out using
Ventana’s Benchmark® XT staining platform. Antigen recovery was conducted under
“standard” conditions with Cell Conditioner 1 buffer (VMSI, cat#950-124). Slides were
incubated with a 1/80 dilution of the stock concentration of the primary anti-PTEN antibody
(Cell Signaling Technology cat#9188) in antibody diluent 95119 (VMSI) or DAKO
(cat#S3022) for 30–60 minutes at 37°C. As a negative control, specimens were incubated
with either a matched IgG rabbit monoclonal antibody or antibody diluent under the same
conditions. Antibody staining was detected using the ultraView™ detection kit (VMSI,
cat#760-500) or the anti-Rb HRP (OmniMap cat#760-4311). Enzymatic detection of antiPTEN antibody was accomplished with a horseradish peroxidase conjugate, followed by
reaction with hydrogen peroxide in the presence of diaminobenzidine and copper sulfate.
The secondary antibody and all chromogen reagents were applied at the instrument’s default
times. Each batch analysis contained positive and negative tumor samples. Both nuclear
and cytoplasmic PTEN staining in tumor and non-tumor tissues was quantified by a
pathologist. Tumor cells were scored manually for PTEN immunoreactivity based on
cytoplasmic and nuclear staining intensity and percentage of tumor cells showing staining
(using a standardized scoring scale of 0 = negative, 1 = weak, 2 = moderate, 3 = strong),
followed by calculating an H-Score for each sample, applying the following formula: H-Score
= 1 x (% weak nuclear staining) + 2 x (% moderate nuclear staining) + 3 x (% strong nuclear
staining). PTEN staining in the non-neoplastic normal vascular endothelium and
extratumoral stromal cells served as the internal positive control for each sample. Tumors
were scored as PTEN-negative when complete absence of nuclear or cytoplasmic staining
(H-Score ≤15) in tumor cells was observed with positive staining in normal cells present in
the same section.
Targeted next-generation sequencing from FFPE tumor sections
Targeted next-generation sequencing was performed at Foundation Medicine, Inc. on
hybridization-captured, adaptor ligation-based libraries using DNA extracted from 4–10
FFPE sections of 4–10 μm thickness from patient tumors. The pathologic diagnosis of each
case was confirmed by a pathologist on H&E-stained slides, and all samples used for DNA
extraction contained a minimum of 20% tumor cell content. Genomic DNA sequencing and
sequence data analysis were performed as described (Lipson et al. Nat Med. 2012;18:382–
384) for 4,604 exons and 48 introns of 296 cancer-related genes, oncogenes and tumor
suppressor genes. The breast cancer cases were sequenced at an average depth of >500X.
Samples were evaluated for genomic alterations including base substitutions, insertions,
deletions, copy number alterations (amplifications and homozygous deletions) and select
gene fusions/rearrangements.
Molecular profiling of circulating tumor DNA
Assessment of phosphoinositide 3-kinase catalytic subunit alpha (PIK3CA) mutation status
at the initiation of pilaralisib or voxtalisib treatment (Cycle 1 Day 1) was performed in cell-free
circulating tumor DNA (ctDNA) obtained from plasma samples using BEAMing assays
(Sysmex Inostics GmbH). Blood was collected in 10 mL BD Vacutainer tubes with K2EDTA
(Becton-Dickinson cat#366643), and plasma prepared within 4 hours of collection by two
serial centrifugations (10 minutes at 1600g and 10 minutes at 3000g) to remove residual cell
contamination, snap-frozen in liquid nitrogen or on dry ice, and stored as frozen aliquots at
−70°C (or below). ctDNA was isolated from plasma and BEAMing assays were conducted
on each sample. The BEAMing assay is a compartmentalized PCR amplification of individual
DNA molecules attached to magnetic beads in water-in-oil emulsions. The mutational status
of DNA bound to beads was determined by allele-specific hybridization of fluorescently
labeled oligonucleotides, and flow cytometry was used to quantify the level of mutant DNA
present in the plasma. Mutations in PIK3CA (8 variants) were analyzed including PIK3CA
mutations in exon 9 (c.1624G>A_p.E542K, c.1633G>A_p.E545K, c.1634A>G_p.E545G,
c.1636C>A _p.Q546K) and exon 20 (c.3129G>T_p.M1043I, c.3139C>T_p.H1047R,
c.3140A>G_p.H1047R, c.3140A>T_p.H1047L).