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Patients

191 women with histological proven breast adenocarcinoma were enrolled in the study
between November 2007 and January 2012 (ClinicalTrials.gov identifier: NCT00959556).
Patients were treated with anthracyclines and/or taxanes based NCT in our institution
(Centre Oscar Lambret, Lille, France). Among them, 118 received FEC100-Taxotere, 46
patients with HER2+ tumors were treated with FEC100-Taxotere-Herceptin, 16 patients
were treated with FEC100 and 11 patients received diverse chemotherapy regimens.

Patient characteristics are summarized in Table 1 for the two main groups. Median age at diagnosis was 46.5 years for patients treated
with FEC100-taxotere and 45.5 years for patients treated with FEC100-taxotere-herceptin.
The median size of the tumors was 30 mm for patients treated with FEC100-taxotere
and 30.5 mm for patients treated with FEC100-taxotere-herceptin. The histoprognostic
grading was established according to Scarff Bloom and Richardson modified by Elston
and Ellis (1998]). FEC100-Taxotere was mostly administrated during 6 cycles (84.7%) and the majority
of patients (82.2%) received 3 cycles of FEC100 followed by 3 cycles of Taxotere.
The median number of treatment cycles of Herceptin per patient was 18 (3–18).

Table 1. Characteristics of patients treated with FEC100-taxotere and FEC100-taxotere-herceptin

Our study only focused on patients treated with FEC100-Taxotere (n = 118), in order
to identify potential predictive polymorphisms in a homogeneous group according to
treatment.

Clinical response assessment

Clinical evaluation of response to therapy was assessed by measuring tumor size (physical
examination and ultrasonography) before NCT, and after 3 and 6 cycles. Change in tumor
size was determined by comparing the tumor size before and after NCT according to
RECIST guidelines, version 1.1 (Eisenhauer et al. 2009]).

Pathological response assessment

Tumor response was assessed by a pathologist and graded according Sataloff et al.
(1995]). Pathological tumor response was used as the gold standard to evaluate treatment
response. On the basis of patients’ pathological response in primary breast site and/or
in the lymph nodes, patients were designated having a pCR when surgical samples showed
total or near-total effect and absence of nodal involvement (TA and NA or NB).

Hormone receptors and HER2 assessment

Immunohistochemistry (IHC) was used for evaluation of oestrogen receptors (ER) (1D5
Dako clone, then SP1 Ventana clone) and progesterone receptors (PR) (1A6 Dako clone,
then PR88 Biogenex clone); to be positive, more than 10% of the tumor nucleus cells
had to be stained. Evaluation of HER2 was performed with IHC (CB11 Biogenex clone,
then HER 485 Dako clone); only tumors classified 3+ were considered positive. All
tumors HER2 2+ with IHC were tested by FISH. Triple negative tumors were defined as
ER and PR negative and HER2 negative by immune histochemistry (IHC) or by FISH in
case of HER2 2+.

DNA extraction

Genomic DNA was extracted from EDTA-treated blood samples (300 µl) using the MagNA
Pure Compact instrument (Roche Diagnostics, Meylan, France) and the MagNA Pure Compact
Nucleic Acid Isolation Kit according to the manufacturer’s instructions.

Genotyping

SNP genotyping was assessed using allelic discrimination with SNPType assays ordered
from Fluidigm. When SNPType assays were not available, TaqMan assays (Life Technologies)
were used instead. Because DNA concentration available was below supplier recommendation,
a Specific Target Amplification was performed to enrich targeted SNP sequences according
to the supplier’s instructions.

Instrumentation and nanofluidic chips

48.48 Dynamic Array used in the present study are nanofluidic chips able to analyze
48 samples with 48 SNP assays on the BioMark platform (Fluidigm). The BioMark system
is used to thermal cycle these nanofluidic chips and image the data in real time.

SNP genotyping using SNPType assays

3 µL of SNPType ASP1/ASP2 (allele specific primer 1 and 2) and 8 µL of SNPtype LSP
were premixed and dilute with 29 µL of DNA suspension buffer to prepare SNPType assay
mix. Each assay (5 µL) comprised 1 µL SNPType assay mix, 2.5µL assay loading reagent
2X (fluidigm) and 1.5 µL DNA-free water. Each sample (6 µL) comprised 3 µL Biotium
fast probe master mix 2X (Biotium), 0.3 µL sample loading reagent 20X (fluidigm),
0.1 µL SNPType reagent 60X (Fluidigm), 0.036 µL ROX 50X (Invitrogen), 0.064 µL DNA-free
water and 2.5µL amplified genomic DNA. Each of the assays (4 µL) and samples (5 µL)
was pipetted into separate inlets in the chip. Amplification was carried out under
the following conditions: 95°C for 5 min, 38 cycles of 95°C for 15 s, 60°C for 45 s,
72°C for 15 s followed by 30 s at 20°C for fluorescence measurement.

SNP genotyping using TaqMan assays

Each assay (5 µL) comprised 2.5 µL assay loading reagent 2X (fluidigm), 0.25 µL ROX
50X (Invitrogen), 1.25 µL SNP Genotyping assay mix 40X (Applied Biosystems) and 1 µL
DNA-free water. Each sample (6 µL) comprised 3 µL TaqMan universal PCR master mix
2X (Applied Biosystems), 0.3 µL sample loading reagent 20X (fluidigm), 0.3 U AmpliTaq
gold polymerase (Applied Biosystems), 0.12 µL DNA-free water and 2.52 µL amplified
genomic DNA. Each of the assays (4 µL) and samples (5 µL) was pipetted into separate
inlets in the chip. Amplification was carried out under the following conditions:
50°C for 2 min, 98°C for 10 min, 40 cycles of 95°C for 15 s, 60°C for 60 s. Fluorescence
was measured at each slice.

Software

The data were analyzed using the BioMark SNP Genotyping Analysis software version
3.1.2 to obtain genotype. Briefly, the software calculates the FAM, VIC or HEX fluorescence
intensities relative to ROX fluorescence background, and then automatically classifies
the samples into three possible genotypes.

Statistical analysis

Clinical and histopathological characteristics were presented as frequencies and percentages
for categorical variables and as medians and range for continuous variables. Associations
between pCR and clinicopathological characteristics were assessed using Khi-2 test
for qualitative variables or Fisher exact test in the case of small counts. Associations
between pCR and continuous variables were performed using Wilcoxon Mann–Whitney test.

After ensuring that Hardy–Weinberg equilibrium was respected, the search of SNP correlated
to pCR was performed by univariate logistic regression (R SNPassoc package). For each
SNP, analyses were done considering genotypes separately or grouped to compare each
homozygous or heterozygous genotype to other ones (only significant results are shown).
Univariate analyses were repeated on subgroups of the population according to ER tumor
status.

Multivariate analysis was performed on whole population combining significant SNP
in a stepwise multivariate logistic regression selecting variables according to the
Akaike Information Criterion (AIC). The model was validated internally on 1,000 random
samples with replacement on the whole dataset. The percentage of times each variable
was selected was extracted. Only those variables which were selected in 80% of models
were retained. The final model was adjusted for ER status.

Results of statistical tests were considered significant at the 5% level. Analyses
were performed using v11.2 Stata software (StataCorp. 2009. Stata Statistical Software:
Release 11. College Station, TX, USA) and v2.15.2 R software (R Core Team 2012]).