Abstract P4-05-16: Quality metric study of real-time targeted massive parallel sequencing (MPS) (ampliseq comprehensive cancer panel (CCP)) and bioinformatics in early breast cancer (EBC) using life technologies ion proton system
Authors: Mainwaring PN , Pyke C , Korbie D , Pheasant M , O'Neill K , Musgrave K , Phillips C , Francis G , Trau M 
Published: August 2014

Abstract
We undertook a prospective quality assurance and metric study in 26 women with early breast cancer (EBC) in order to identify issues that may interfere with real-time integration of massive parallel sequencing (MPS) with clinical and pathology parameters for clinical decision making.
Pre-operatively, patients underwent formal informed consent process with the aid of bespoke multimedia (iPad) technology to educate patients on a) the logistical aims of the trial b) somatic and hereditary genomics c) privacy and off target (non-cancer) issues.
On Day 1, seven patients (27%) underwent WLE & SNB, 17 (65%) WLE & AXLND, 2 (7%) Mastectomy & SNB.
Samples (tumour and normal) were collected fresh into Qiagen PaxGene Tissue Container preserving both morphology and biomolecules of the sample with lysis of tissues overnight.
Day 2&3; DNA extraction was performed using the Qiagen DSP kit over 1 hour.
DNA MPS CCP was performed on Life technologies Proton P1 chip using in-house modification of manufacturers protocol. The average time taken to run CCP includes 2 days library preparation; 4 hour sequencing run, 8 hours primary analysis for generating read alignments and sequencing files. The Ampliseq protocol required modifications with respect to template input amount and cycling parameters in order to increase the fidelity of library preparation. Limitations in sequencing depth and read qualities with the current technologies lend themselves to approximately 5-6 samples/sequencing run/day if 500x read depth per amplicon is desired.
Days 4-6 manually curated bioinformatics against publicly available mutation databases (e.g. COSMIC, ENSEMBL etc.)
Day 7 communication of results
Molecular Pathology results
The median age was 57, (32-84), 18 (72%) were postmenopausal. Histopathology invasive ductal 22 (84%), invasive lobular 2 (8%) metaplastic 2 (8%).
TNM Staging: T1b 5 (19%), T1c 9 (35%) T2 12 (46%); N0 23 (88%), N1mi 1 (4%), N1a 1 (4%), N2a 1 (4%).
Hormone receptors antibodies: ER (Roche SP1) Strong 20 (77%), Moderate 1 (4%), Weak 1 (4%), Negative 4 (15%); PR (Roche 1E2) Strong 11 (42%), Moderate 10 (38%), Weak 0 (0%), Negative 5 (19%); HER2 IHC (Ventana 4B5) Strong 1 (4%), Moderate 6 (23%), Weak 6 (23%), Negative 13 (50%); HER2 SISH (Ventana inform single probe); 1 SISH positive (mean copy number 29.6); 4 patients (15%) triple negative.
Molecular aberrations identified included oncogenes TP53 2 (8%), PIK3CA 12 (46%) HRAS 2 (8%), AKT1 1 (4%); tumour suppressor genes MLL3 2 (8%), MSH6 1 (4%), ARID1A 1 (4%), and in the germline in tumour suppressor genes EPHB6 1 (4%) and LTK 1 (4%).
Significant technical and bioinformatic challenges were encountered during the development of this metric study including Ampliseq protocol modification and parameter tuning for variant calling algorithm.
In summary, many challenges face the clinical laboratory developing high throughput MPS using desktop MPS. To date this is the first publication of the new Ion Proton P1 chips in development for real-time clinical decision making. Copy-number variation (CNV), RNA-Seq, Methyl-Seq data will be available for presentation at San Antonio.
Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-05-16.