P-0197 - Comparison of a new multiplexed multigene approach with pyrosequencing in diagnostic molecular biology of colorectal cancer (CRC)

Date 28 June 2014
Event World GI 2014
Session Poster Session
Topics Colon Cancer
Rectal Cancer
Pathology/Molecular Biology
Presenter Giovanna De Maglio
Citation Annals of Oncology (2014) 25 (suppl_2): ii14-ii104. 10.1093/annonc/mdu165
Authors G. De Maglio1, E. Ongaro1, L. Ferrari1, G. Aprile2, G. Fasola3, S. Pizzolitto1, S. Cernic1, L. Pagani1
  • 1University Hospital Udine, Udine/IT
  • 2University Hospital of Udine, Udine/IT
  • 3University Hospital S. Maria della Misericordia, Udine/IT



In the last years, our understanding of the biology of colorectal cancer (CRC) has improved significantly. Studies on the molecular pathology of CRC have demonstrated that genomic alterations on EGFR gene pathway have a clear predictive and prognostic role. As cancer research promotes characterization of individual diseases based on molecular profiles, it is becoming essential to cross-examine multiple genes for each tumour. Both KRAS and NRAS mutational status analysis have been introduced in the clinical practice to select patients suitable for anti-EGFR monoclonal antibodies. Patients harboring BRAF gene mutations are characterized by a worse prognosis compared to wild-type BRAF patients. PIK3CA mutations may also impact on outcome results. To optimize the management of CRC patients, however, a more profound knowledge of tumor molecular oncology is crucial for the clinicians. This study compare the use of a multiplexed multigene mutation detection panel using Sequenom MassARRAY® Platform with pyrosequencing.


Formalin-fixed and paraffin-embedded sample of 101 cases were previously tested for mutational status of KRAS and BRAF by pyrosequencing with PyroMarkTMQ96 ID instrument (Qiagen, Germany) with CE-IVD commercially available kits Anti-EGFR MoAb response (Diatech Pharmacogenetics, Italy). All cases have been blinded rested on Sequenom MassARRAY® System with CE-IVD Myriapod® Cancer status kit (Diatech Pharmacogenetics, Italy) on Sequenom MassARRAY® System (Sequenom, Inc. California), a target method designed to investigate more than 200 mutations on KRAS, BRAF, NRAS and PIK3CA genes. Univariate analysis was used to assess concordance.


Concordance rate between the two methods was 98% (p < 0.05). In only 2 samples (1.8%) results were discordant, and Sequenom® allowed to discover mutations (KRAS p.G12V and KRAS p.G12R) that were indeterminate by pyrosequencing. Sequenom® test needs 40 ng of DNA for 4 gene assessment, compared to the requested amount of at least 250 ng for KRAS/BRAF/NRAStest on pyrosequencing. The simultaneously detection of four genes in the same analytic session, even requiring sample batching, provided a high throughput flexibility saving time. Comparing the time to molecular diagnosis, pyrosequencing approach allowed KRAS/BRAF/NRAS profile in no less than 4 working days, while Sequenom system was able to guarantee KRAS/BRAF/NRAS/PIK3CA panel in 2 days.


Sequenom MassARRAY® System is a time-saving target method for detection of known mutations, whose concordance with pyrosequencing analysis is almost prefect. Also, Sequenom® System may be helpful when only small endoscopic/cytological samples are available for analysis in order to obtain accurate results, with a sensitivity up to 2.5%, using a smaller amount of DNA in a single analytical run.