DETAILED METHODS
Regulatory approval and registration of the clinical studies
Clinical protocols were approved by local regulatory authorities and were also included in the Australian New Zealand Clinical Trials Registry (ANZCTR) and allocated the following Registration Numbers: ACTRN12611000506998 (HE10/97) and ACTRN12609001036202 (HE10/00). All patients signed a study-specific written informed consent before randomization, which in addition to giving consent for the trial allowed the use of their biological material for future research purposes.
Patient subtype classification (immunophenotypical)
Patients were classified, based on ER, PgR, HER2 and Ki67 protein expression, as luminal A (ER-positive and/or PgR-positive, HER2-negative and Ki67low), luminal B (ER-positive and/or PgR-positive, HER2-negative and Ki67high), luminal-HER2 (ER-positive and/or PgR-positive and HER2-positive), HER2-enriched (ER-negative, PgR-negative, HER2-positive) and triple-negative breast cancer (TNBC, ER-negative, PgR-negative, HER2-negative). HER2 status was considered to be positive if HER2 was amplified (ratio >2.2 or gene copy number >6) by FISH and/or a HER2 score of 3+ was obtained by IHC.
Tissue microarray (TMA) construction
FFPE tumor tissue samples (paraffin blocks) were collected retrospectively in the first trial (HE10/97) and prospectively in the second (HE10/00). New representative hematoxylin-eosin stained sections from the tissue blocks were reviewed by two pathologists and the most representative tumor areas were marked for the construction of the ΤΜΑ blocks with the use of a manual arrayer (MTA-1, Beecher Instruments, San Prairie, WI). Each case was represented by 2 tissue cores, 1.5 mm in diameter, obtained from the most representative tumor areas of primary invasive and in 9.6% of cases from synchronous axillary lymph node metastases and re-embedded in 51 microarray blocks. Each TMA block contained tissue cores from the original tumor tissue blocks as well as cores from various neoplastic, non-neoplastic and reactive tissues, serving as assay controls. Cases not represented, damaged or inadequate on the TMA sections were re-cut from the original blocks and these sections were used for protein and gene analysis.
Immunohistochemistry (IHC)
IHC for ER (clone 6F11, Leica Biosystems, Newcastle, Upon Tyne, UK), PgR (clone 1A6, Leica), HER2 (A0485, polyclonal, Dako, Glostup, DK), Ki67 (MIB1, Dako), and EGFR (clone 31G7, Invitrogen, Camarillo, CA) was performed on serial 2.5 μm thick sections, using the Bond MaxTM and Bond IIITM autostainers (Leica Microsystems, Wetzlar, Germany), as previously described in detail1. IHC was also performed with antibodies against PTEN, clone 6H2.1, Dako, Glostrup, DK, at dilution 1:200, for 30 min; phosphorylated Akt1/2/3 at threonine 308, sc-16646-R, Santa Cruz, Santa Cruz, CA, at dilution 1:1000 overnight at 4OC; phosphorylated Akt1/2/3 at serine 473, clone 736E1, Cell Signaling Technology (CST), Danvers, MA, at dilution 1:150, overnight at 4OC; and phosphorylated mTOR at serine 2448, clone 49F9, CST, at dilution 1:30 for 20 min.
The sections of the TMA blocks were stained in one run for each antibody, at the Laboratory of Molecular Oncology of the Hellenic Foundation for Cancer Research, Aristotle University of Thessaloniki School of Medicine. All cases were also stained for vimentin and cytokeratin 8/18, which were used as control stains for tissue immunoreactivity and fixation, as well as identification of tumor cells. Tissue samples negative for the above antibodies were excluded from the study. The evaluation of all IHC sections was done by experienced in breast cancer pathologists, blinded as to the patients’ clinical characteristics and survival data.
Interpretation of the IHC results
ER, PgR, HER2, was evaluated according to the of ASCO/CAP guideline recommendations2,3. Ki67, protein expression was evaluated using the recent proposed criteria4. Sections for EGFR were considered positive when ≥1% of the tumor cells had membranous staining above the background level and the intensity of EGFR reactivity was scored as +1 (mild), +2 (moderate) and +3 (strong)5. PTEN protein (cytoplasmic, nuclear or both) was evaluated according to a staining intensity scale from 0 (negative, no staining) to 3 (intense staining) and percentage of stained cells. Positive tumors were defined as having greater than 10% of cells staining positive6.
In the two phosphorylated forms of p-Akt we evaluated the staining in all cell compartments. The p-AktSer473 was considered positive when the expression was at least 1 (mild) in >1% of tumor cells7. For p-AktThr308, positive cases were considered those that had at least intensity of 2 in >10% of tumor cells8. Apart expression in tumor cells the expression of two p-Akt’s was also evaluated and for the tumor stroma. The phosphorylated form mTOR at Ser2448, was defined as positive if at least mild cytoplasmic staining was detected in >1% of tumor cells9.
Fluorescence in situ hybridization (FISH)
FISH analysis of HER2 gene status was performed using the ZytoLight® SPEC HER2/TOP2A/CEN17 triple color probe (Zytovision, Bremerhaven, Germany), as previously described10. TOP2A gene amplification data was not included for the purposes of the present study, since this marker is not used for breast cancer subtyping.
Twenty tumor nuclei were counted according to Press et al11. The HER2 gene was considered to be amplified when the ratio of the gene probe/centromere probe was >2.23, or the HER2 copy number was >612. HER2 status was considered to be positive if HER2 was amplified (ratio >2.2 or copy number >6) by FISH and/or a HER2 score of 3+ was obtained by IHC.
Single nucleotide polymorphism (SNP) genotyping for PIK3CA mutations
DNA was extracted from FFPE whole tissue sections or macrodissected tissue fragments containing >70% tumor cells, using a fully automated isolation method based on silica-coated magnetic beads (Versant Tissue Preparation Reagents, Siemens Healthcare Diagnostics, Tarrytown, NY) in combination with a liquid handling robot, as previously described13. Mutation testing for PIK3CA E542K and E545K (exon 9) and H1047R (exon 20) was accomplished with custom Taqman-MGB-SNP genotyping assays (duplex qRT-PCR for the detection of control DNA and mutant target in the same reaction). Samples were normalized at 50ng/10ul reaction and were run in duplicates under default conditions for Allelic Discrimination in an ABI7500 sequence detection system equipped with the SDS v1.4 software (Applied Biosystems). Sequencing validation was performed in selected cases (n=4 for each assay).
References
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