Supplementary methods{Level 1 heading}
Assessment of tumor response in the patient{Level 2 heading}
For each line of chemotherapy, the patient response under treatment was characterized.
Metabolic response was assessed according to Positron Emission Tomography Response Criteria in Solid Tumors (PERCIST) criteria [1]. In summary, partial metabolic response (PMR) is defined by a reduction in SUVmax (maximum standardized uptake value within the region of interest) of at least 30%, with no new lesions. Complete metabolic response (CMR) corresponds to the disappearance of all lesions in the blood-pool background. Progressive metabolic disease (PMD) is defined by an increase in SUVmax greater than 30% or the appearance of new fluoro-deoxyglucose (FDG)-avid lesions. Stable metabolic disease applies when criteria for other categories (CMR, PMR, or PMD) are not met.
For each line of chemotherapy, time-to-progression was defined as the time between initiation of treatment and diagnosis of disease progression. 18FDG (5 MBq/kg; not exceeding 500 MBq) was injected intravenously 60 minutes before data were acquired on a Philips Gemini XL positron emission tomography/computed tomography (PET/CT) scanner (Philips, Eindhoven, The Netherlands). CT data were acquired first (120 kV; 100 mAs; no contrast enhancement). PET three-dimensional (3D) data were acquired with 2 minutes per bed position, and images were reconstructed by using a 3D row-action maximum likelihood algorithm.
PET/CT images were interpreted by a nuclear medicine physician (LV) blinded to the patient’s record. 18FDG uptake was expressed as SUV. A 3D region of interest was drawn around the lesions, and SUVmax was measured. SUVmax values of the lesions with the highest uptake were recorded and used for the study analysis (five target lesions were assessed). SUVmax of the liver was also recorded as a control value. The change in SUVmax at each evaluation was expressed as ΔSUVmax (percentage) = 100 × (cycle n SUVmax – cycle (n-1) SUVmax) / cycle n-1 SUVmax. Appearance of new lesions was also recorded.
Breast cancer sample and gene expression profiling{Level 2 heading}
Five samples were obtained during the procedure of imaging-guided biopsy of the lymph node metastasis at the time of relapse, before any medical treatment. Informed written consent was obtained from the patient.
Among the five tumor samples, (a) two were formaldehyde-fixed and paraffin-embedded for histological analyses, (b) two were immediately snap-frozen in liquid nitrogen and stored in Hôpital-Saint-Louis Tumorbank for molecular analysis, and (c) one was set aside in culture medium for xenograft.
Total RNA was extracted from the frozen tumor sample by using an RNeasy-Mini-Kit (Qiagen, Courtaboeuf Cedex, France), quantified on NanoDrop (LabTech, Palaiseau, France) and qualified on Bio-Rad Experion™ Automated-Electrophoresis-Station (Bio-Rad, Marnes-la-Coquette, France). Transcriptomic analyses were performed by using MiltenyiBiotec Microarray service (MiltenyiBiotec, Bergisch Gladbach, Germany). A linear T7-based amplification step was performed from 0.5 µg of all RNA samples. To produce Cy3-labeled cRNA, the RNA samples were amplified and labeled by using an Agilent-Quick-labeling kit (Agilent Technologies, Santa Clara, CA, USA). The yields of cRNA and the dye-incorporation rate were measured with an ND-1000 Spectrophotometer (NanoDrop, LabTech). Hybridization was performed according to the Agilent 60-mer oligo-microarray processing protocol: 1.65 µg of Cy3-labeled cRNA was hybridized overnight at 65°C to Agilent-Whole-Human-Genome-Oligo-Microarrays 4x44K, and fluorescence signals were detected by using Agilent’s Microarray-Scanner. Agilent-FE-Software determined feature intensities, and quantile normalization was performed with the Agi4x44PreProcess R package. Subsequent analyses were carried out with R 3.01 software (Foundation for Statistical Computing, Vienna, Austria) and based on log2 single-intensity expression data. Classification was provided by correlating gene expression profiles with the centroids for each of the six TNBC subtypes described by Lehmann and colleagues [2] and to Parker centroids for PAM50 classification [3,4].
Gene profile validation{Level 2 heading}
Sequencing of KRAS was performed as previously reported [5].
Breast cancer sample and xenografts{Level 2 heading}
The University Institute Board Ethics Committee for experimental animal studies approved this study (#2012-15/728-0115). Nude mice, purchased from Janvier (Centre-Elevage-Janvier, Palaiseau, France), were maintained in specific pathogen-free animal housing (IUH, Paris). After imaging-guided human tumor biopsies had been performed, one sample was transported in RPMI-1640 and subcutaneously grafted in 6-week-old NMRI-nude mice, under xylasin (10 mg/kg)/ketamin (100 mg/kg) anesthesia.
A successful engraftment of the metastastic samples was observed after 3 weeks, a clinical score being recorded daily for the mice and tumor growth being measured in two perpendicular diameters with a caliper. Tumor volumes were calculated as V=L×l²÷2, L being the larger diameter (length), l the smaller (width). After mouse euthanasia, the tumor was resected, cut into small pieces of 1 mm3, and grafted again in 50 other nude mice. When tumors reached a volume of 300 mm3 (n = 5 mice per treatment group), after 2 to 4 weeks, the mice were treated over 1 month with eight regimens of chemotherapy (see Supplementary Table for details). Ultrasonography was performed twice a week on treated and untreated mice with an AplioXT ultrasonograph (Toshiba, Tokyo, Japan) to assess tumor response.
Abbreviations{Level 1 heading}
3D, three-dimensional; CMR, complete metabolic response; CT, computed tomography; FDG, fluoro-deoxyglucose; PERCIST, Positron Emission Tomography Response Criteria in Solid Tumors; PET, positron emission tomography; PMD, progressive metabolic disease; PMR, partial metabolic response; SUVmax, maximum standardized uptake value within the region of interest.
References{Level 1 heading}
1. Wahl RL, Jacene H, Kasamon Y, Lodge MA: From RECIST to PERCIST: evolving considerations for PET response criteria in solid tumors. J Nucl Med 2009, 50 Suppl 1:122S-150S.
2. Lehmann BD, Bauer JA, Chen X, Sanders ME, Chakravarthy AB, Shyr Y, Pietenpol JA: Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies. J Clin Invest 2011, 121:2750-2767.
3. Parker JS, Mullins M, Cheang MC, Leung S, Voduc D, Vickery T, Davies S, Fauron C, He X, Hu Z, Quackenbush JF, Stijleman IJ, Palazzo J, Marron JS, Nobel AB, Mardis E, Nielsen TO, Ellis MJ, Perou CM, Bernard PS: Supervised risk predictor of breast cancer based on intrinsic subtypes. J Clin Oncol 2009, 27:1160-1167.
4. genome.unc.edu [https://
genome.unc.edu/pubsup/breastGEO/pam50_centroids.txt].
5. Blons H, Rouleau E, Charrier N, Chatellier G, Côté JF, Pages JC, de Fraipont F, Boyer JC, Merlio JP, Morel A, Gorisse MC, de Cremoux P, Leroy K, Milano G, Ouafik L, Merlin JL, Le Corre D, Aucouturier P, Sabourin JC, Nowak F, Frebourg T, Emile JF, Durand-Zaleski I, Laurent-Puig P; MOKAECM Collaborative Group: Performance and cost efficiency of KRAS mutation testing for metastatic colorectal cancer in routine diagnosis: the MOKAECM study, a nationwide experience. PLoS One 2013, 8:e68945.
{2 Column Table}
Supplementary Table. Administration of drugs in xenografted mice
Drug / Dose (mg/kg) / Mode of administration / Weekly frequency of administrationEpirubicin / 1 / IP / Once
Cyclophosphamide / 30 / IP / Twice
Bevacizumab / 5 / IP / Three times
Paclitaxel / 20 / IP / Twice
Cetuximab / 10 / IP / Twice
Cisplatin / 3 / IP / Once
Oxaliplatin / 1 / IP / Twice
IP, intraperitoneal.