Sample selection
Breast cancers diagnosed as HER2-positive but showing HER2 heterogeneous overexpression were selected and retrieved from the authors’ institutions and re-reviewed by three pathologists (AG, AV-S and JSR-F). In brief, over 250 HER2-positive breast cancers diagnosed at Institut Curie, Paris, France between 2005 and 2008 and treated with conservative surgery as a first step of treatment were reviewed at Institut Curie and cases showing a heterogeneous overexpression pattern for HER2 defined as >10% but <100% of cells displaying HER2 overexpression in the form of strong, complete membrane staining were identified. Furthermore, cases with similar staining patterns were obtained from four French Comprehensive Cancer Centers (Centre Georges François Leclerc, Dijon; Institut Claudius Regaud, Toulouse; Centre Jean Perrin, Clermont-Ferrand; Centre Oscar Lambret, Lille), and from Hospital Israelita Albert Einstein, São Paulo, Brazil, and submitted for further review at Institut Curie, Paris, France. Analysis of human samples was performed in accordance with the French Bioethics Law 2011-814, the French National Institute of Cancer (INCa) Ethics Charter and after approval by the Institut Curie Review Board and Ethics committee (Comité de Pilotage du Groupe Sein; project 'Repertoire des alterations genetiques somatiques dans les adenocarcinomes mammaires avec heterogeneite du statut HER2', final version approved on 18 June 2013). Written consent was obtained from patients whose samples were subjected to massively parallel sequencing. Cases were anonymized prior to genomic profiling and massively parallel sequencing analyses. This study is compliant with the Declaration of Helsinki.
Immunohistochemistry and chromogenic in situ hybridization
Formalin-fixed, paraffin-embedded sections of the selected HER2 heterogeneous breast cancers were cut at 3 μm, and immunohistochemistry performed for ER, PR and Ki67 using the antibodies and antigen retrieval methods described in Duprez et al. [56]. For confirmation, tumor sections were stained for HER2 using the HercepTest (Dako, Glostrup, Denmark) [57]. Scoring was performed by two pathologists (AV-S and JSR-F) according to the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines [1,25]. Only cases with distinct areas of HER2 3+ positivity with adjacent areas of HER2 1+ positivity or absent HER2 overexpression were included (n = 41). Cases with admixed levels of HER2 overexpression, including HER2 2+ (that is, equivocal), were excluded. In addition to the local FISH analyses performed, HER2 gene amplification was confirmed by CISH, which allowed for the assessment of HER2 gene copy number status and its distribution in the neoplastic tissues. CISH was performed using the ZytoDot 2C SPEC HER2/CEN 17 Probe Kit (Zytovision GmbH, Bremerhaven, Germany) or the HER2 CISH pharmaDX kit (Dako) according to the manufacturers’ instructions. HER2 gene amplification was defined according to ASCO/CAP guidelines [1] and assessed by three pathologists (AG, AV-S and JSR-F). Tumors were graded according to the Nottingham grading system [58] by three pathologists (AG, AV-S and JSR-F).
Microdissection and DNA extraction
Of the cases reviewed and selected, 13 breast cancers with heterogeneous HER2 protein overexpression and HER2 gene amplification by central IHC and CISH were amenable to microdissection, as HER2-positive and HER2-negative areas were sufficiently discrete. The HER2-positive and HER2-negative components were microdissected either on a PixCell II laser capture microdissector (Arcturus, Life Technologies, Paisley, UK) into separate tubes from 8 μm-thick representative tissue sections stained with HercepTest for guidance, or using a sterile needle under a stereomicroscope in selected cases as previously described [59]. DNA was extracted from the microdissected HER2-positive and HER2-negative components using DNeasy Blood and Tissue kit (Qiagen, Crawley, UK), and quantity was assessed using a PicoGreen assay (Life Technologies, Paisley, UK).
Microarray-based comparative genomic hybridization
DNA obtained from the microdissected HER2-positive and HER2-negative components of 13 HER2 heterogeneous breast cancers was subjected separately to aCGH, using a 32K bacterial artificial chromosome (BAC) array platform with 50 kb resolution [56,57]. This platform has been shown to be as robust as, and to have comparable resolution with, high-density oligonucleotide arrays [60-62] and to perform well with DNA extracted from formalin-fixed paraffin-embedded tissue samples. DNA labeling and hybridization, image acquisition and data analysis were performed as previously described [56,57] (Additional file 19). The aCGH analysis script and code are available in Additional file 20.
Whole exome sequencing and targeted sequencing
Microdissected frozen samples of the HER2-positive component, the HER2-negative component and the matched normal tissue from three cases (T6, T11 and T12) were subjected to whole exome sequencing (Agilent SureSelect, Santa Clara, CA, USA) on an Illumina Genome Analyzer IIx or Illumina HiSeq2000 platform to a median coverage of 81× (Additional files 8 and 19). Candidate somatic variants with mutant allele frequencies >15% identified by whole exome sequencing in at least one component were subjected either to deep re-sequencing on an Ion Torrent platform (Life Technologies), or to targeted capture massively parallel sequencing on an Illumina HiSeq2000 (Additional file 19). Clonal mutation frequencies were inferred using ABSOLUTE [47]. In addition, five of the twelve cases subjected to aCGH profiling had sufficient DNA from tumor and normal tissues to be subjected to custom 273 gene paired-end massively parallel targeted sequencing on an Illumina HiSeq2000 essentially as previously described [48]. Details of the coverage and depth obtained in each component of these cases are described in Additional file 8. The strategy for the classification of mutations according to their pathogenicity is outlined in Additional file 19.
Sanger sequencing
Sanger sequencing of exons 1 to 11 of TP53 was performed in the HER2-positive and HER2-negative components of all HER2 heterogeneous breast cancers as previously described [48] (for primer sequences, see Additional file 21). A perfect agreement between the results of TP53 Sanger sequencing and TP53 mutation status as defined by massively parallel sequencing was observed for the cases analyzed.
Cell lines
MCF10A, MCF12A, MCF7, T47D, BT474, HEK293T and NIH3T3 cells were purchased from the American Type Culture Collection (ATCC), authenticated by short tandem repeat profiling as previously described [63], and tested for mycoplasma infection using a PCR-based test (ATCC). Culture conditions are described in Additional file 19.
Vector construction, mutagenesis, transformation and plasmid preparation
The human ERBB2 (NM_004448) cDNA ORF clone pCMV6-ERBB2::Myc-DDK was purchased from Origene (RC212583, Rockville, MD, USA), and the I767M mutation introduced using the GeneArt Site Directed Mutagenesis Kit (Life Technologies) following the manufacturer’s recommendations (pCMV6-ERBB2(I767M)::Myc-DDK). ERBB2 (HER2) wild-type and mutant (I767M) open reading frames were cloned into the pCMV6-TagRFP vector to generate pCMV6-ERBB2::TagRFP and pCMV6-ERBB2(I767M)::TagRFP plasmids, respectively, and into the pLenti-EF1a-GFP-2A-Puro vector (LV067, ABM, Richmond, BC, Canada), to generate the pLenti-ERBB2 and pLenti-ERBB2(I767M) lentiviral plasmids, as previously described [45] (Additional file 19). BRF2 and DSN1 open reading frames were amplified from total RNA derived from a healthy donor using SuperScript III First Strand Synthesis System and Platinum Taq polymerase High Fidelity (Life Technologies), and cloned into the pCMV6-ZsGreen vector to generate pCMV6-BRF2::ZsGreen and pCMV6-DSN1::ZsGreen plasmids, respectively, and into the pLenti-EF1a-GFP-2A-Puro vector (LV067, ABM), generating the pLenti-BRF2 and pLenti-DSN1 lentiviral plasmids, respectively. Sanger sequencing was used to confirm the reading frames of the wild-type ERBB2, the I767M mutant ERBB2, and wild-type BRF2 and DSN1. Primer sequences are available in Additional file 21.
Transfections of mammalian cells and analysis of transgene expression
Transfections of empty vector, wild-type ERBB2, BRF2 and DSN1, and I767M mutant ERBB2 were performed essentially as previously described [45] (Additional file 19). The expression of transgenes in stable clones for DSN1 and BRF2 was evaluated at the mRNA level by qualitative and quantitative RT-PCR (Additional files 7 and 19), given that antibodies producing satisfactory western blot results were not available. The expression of wild-type and I767M mutant HER2 proteins was confirmed by western blot (see below). The expression of transgenes from pCMV-ZsGreen/TagRFP-derived plasmids was visually evaluated 48 hours after transfection using a Nikon Eclipse Ti fluorescence microscope.
Confocal microscopy for BRF2 and DSN1 subcellular localization
Cells expressing BRF2-ZsGreen and DSN1-ZsGreen, TagRFP and ZsGreen (control) proteins grown on coverslips were fixed for 15 minutes in 10% buffered formalin, washed with 1× phosphate buffered saline (PBS), counterstained with 300 nM 4',6-diamidino-2-phenylindole (DAPI; Life Technologies) for 2 minutes, and mounted using ProLong Gold Antifade Reagent (LifeTechnologies). After 24 hours, fluorescence images were acquired using a Leica TCS SP5-II Upright microscope.
Growth curves
T47D and MCF7 cells stably expressing HER2 wild-type, HER2(I767M) and vector control (T47D, 1,000 cells/well and MCF7, 500 cells/well) were seeded in the corresponding normal growth medium in 96-well plates in triplicate as previously described [45] (Additional file 19). Growth curves were plotted and analyzed (multiple t-tests, corrected for multiple comparisons using the Holm-Šídák method, alpha: 0.05) using GraphPad Prism v_6.0c (GraphPad Software, Inc., La Jolla, CA, USA).
Protein fractionation
Cytoplasmic and membrane/organellular enriched protein fractions from MCF7 and T47D cells expressing HER2 wild-type and HER2(I767M) proteins and vector control were prepared using a Cell Fractionation Kit (Cell Signaling Technologies, Danvers, MA, USA) following the manufacturer’s protocol. To determine the efficiency and purity of the cell fractionation, the separated subcellular fractions were assayed by western blotting using antibodies against MEK1/2 (cytoplasm) and AIF (membrane/organellular) (Cell Fractionation Antibody Sampler Kit, Cell Signaling Technologies), and against HER2 as previously described [45].
Western blotting
Standard western blotting was conducted as previously described [63]. Antibodies and dilutions are described in Additional file 19. Quantification of conjugated secondary antibodies and analysis were performed using the Image Studio Software from LI-COR (LI-COR Biosciences, Lincoln, NE, USA).
Foci formation
NIH3T3 cells expressing HER2 wild-type, HER2(I767M), BRF2 and DSN1 protein, and empty vector control cells were seeded at 5 × 105 cells density in six-well culture dishes in full media without penicillin/ streptomycin for up to 21 days, then fixed with methanol and stained with 0.5% (w/v) crystal violet. Photomicrographs were taken using a Nikon DS5000 digital camera at day 21 for empty vector, BRF2 and DSN1, and using the EVOS XL Imaging System (Life Technologies) at days 5 and 12 for HER2 wild-type, HER2(I767M) and empty vector. All experiments were performed in triplicate. Foci were counted at the end of the assays using Image J and analyzed using GraphPad Prism v_6.0c (unpaired Student’s t-test, two-tailed).
Anchorage-independent growth
Anchorage-independent transformation assays were performed using the Cell Biolabs CytoSelect 96-well Cell Transformation Assay (colorimetric, Cell Biolabs, San Diego, CA, USA) following the manufacturer’s instructions. Briefly, MCF10A cells stably expressing HER2 wild-type, HER2(I767M), BRF2 and DSN1 proteins, as well as vector control cells, were incubated in a proprietary semisolid agar media for 8 days before being solubilized, transferred and detected by the provided MTT Solution (570 nm) using the Victor X4 Multimode Plate Reader (PerkinElmer, Waltham, MA, USA). Assays were performed in quadruplicate reactions. For colony counting, soft-agar cultures were set up in triplicate as described above for i) MCF10A cells (stable for HER2 wild-type, HER2(I767M), BRF2, DSN1 and empty vector; transient for HER2 wild-type, HER2(I767M), HER2(Y835F), HER2(V777L) and empty vector), ii) NIH3T3 cells (stable for HER2 wild-type, HER2(I767M) and empty vector; transient for HER2 wild-type, HER2(I767M), HER2(Y835F), HER2(V777L) and empty vector), and iii) MCF12A cells (transient for HER2 wild-type, HER2(I767M), HER2(Y835F), HER2(V777L) and empty vector). Colony number and size were documented at day 9 using the phase contrast EVOS XL Imaging System (Life Technologies). Colonies were counted in Image J and colony size was determined in MetaMorph Image Analysis software (Molecular Devices, Sunnyvale, CA, USA). Analyses were carried out using GraphPad Prism v_6.0c.
ERBB2-TagRFP and ERBB2(I767M)-TagRFP immunoprecipitation and tyrosine kinase assay
Forty-eight hours post-transfection with pCMV6-ERBB2::TagRFP, pCMV6-ERBB2(I767M)::TagRFP and the vector control, HEK293T cells were treated with 10 ng/ml of human neuregulin-1 (hNRG-1, Cell Signaling Technologies) for HER2 pre-activation or vehicle (20 mM citrate, pH 3.0) for 15 minutes. TagRFP antibody (Evrogen, Farmingdale, NY, USA) was crosslinked to magnetic beads using the PierceTM Crosslink Magnetic IP/Co-IP Kit (Thermo Scientific, Somerset, NJ, USA) following the manufacturers’ recommendations. On the day of preparation, 600 μl of protein lysates at 1 mg/ml were pre-cleared using 75 μl of washed protein A/G magnetic bead slurry and incubated for 1 hour at 4°C. Triplicates of 200 μl (1 mg/ml) of the pre-cleared lysates were then incubated with the equivalent of 5 μg of TagRFP antibody conjugated to magnetic beads under gentle rocking overnight at 4°C. The magnetic beads were then pelleted by placing the tubes in a magnetic separation rack. The magnetic bead pellets were washed five times with 1 ml of ice-cold M-PER Mammalian Protein Extraction Reagent supplemented with Halt Protease and Phosphatase inhibitors cocktail (Thermo Scientific). All steps were carried out at 4°C. Tyrosine kinase activity was evaluated using the ADP Hunter HS Assay (DiscoveRx, Fremont, CA, USA) and the Tyrosine Kinase Assay Kit (colorimetric detection, Millipore, Billerica, MA, USA) essentially as previously described [45] (Additional file 19).
Three-dimensional matrigel cultures
MCF10A and MCF12A cells expressing HER2 wild-type, HER2(I767M), BRF2 and DSN1 proteins, as well as vector control cells, were seeded on top of growth factor-reduced reconstituted basement membrane (Matrigel, BD Biosciences, San Jose, CA, USA) and analyzed essentially as previously described [45] (Additional file 19).
Data availability
aCGH data have been deposited into the NCBI Gene Expression Omnibus under the accession GSE67908. The R code for analysis of the aCGH data is deposited on GitHub [64]. Whole exome data have been deposited into the NCBI Sequence Read Archive under the accession SRP049005.