Marie Rouyer

Validation of a Phosphoprotein Array Assay for Characterization of Human Tyrosine Kinase Receptor Downstream Signaling in Breast Cancer

BACKGROUND Human epidermal growth factor receptor (HER) downstream signaling kinases have important effects on tumor response to anti-HER monoclonal antibodies and tyrosine kinase inhibitors. We validated an assay that uses phosphoprotein arrays for measurement of HER downstream signaling functionality in breast carcinomas. METHODS Using the Bio-Plex(R) phosphoprotein array (BPA), we performed multiplex immunoanalysis to investigate the expression of phosphorylated epidermal growth factor receptor and phosphorylated HER downstream signaling proteins (phosphorylated protein kinase B, phosphorylated glycogen synthase kinase -3beta, phosphorylated P70 ribosomal protein S6 kinase, and phosphorylated extracellular signal regulated kinase 42/44) in 49 frozen specimens of ductal infiltrating breast carcinoma taken at diagnosis. BPA was cross-validated with Western blot analysis. Sample size, homogenicity, tumor content, protein extraction, and monoclonal antibody detection were in accordance with optimized standard operating procedures. RESULTS Linear regression showed significant quantitative correlations between BPA and Western blot, with regression coefficient values of 0.71-0.87 (P < 0.001). BPA intra- and interassay CVs were <17% and 15%, respectively. Compared to limits of detection established by using the mean + 3SD of 10 blanks, large variations of phosphoprotein expression, up to several hundred-fold, were observed among the 49 tumor specimens. CONCLUSIONS Our results validate the use of the multiplex phosphoprotein array assay in human clinical tumor specimens. Further prospective evaluation is warranted to investigate the use of HER downstream signaling phosphoproteins as predictive and/or surrogate markers for clinical response to anti-HER targeted therapy.

Optimization of routine KRAS mutation PCR-based testing procedure for rational individualized first-line-targeted therapy selection in metastatic colorectal cancer

KRAS mutation detection represents a crucial issue in metastatic colorectal cancer (mCRC). The optimization of KRAS mutation detection delay enabling rational prescription of first‐line treatment in mCRC including anti‐EGFR‐targeted therapy requires robust and rapid molecular biology techniques. Routine analysis of mutations in codons 12 and 13 on 674 paraffin‐embedded tissue specimens of mCRC has been performed for KRAS mutations detection using three molecular biology techniques, that is, high‐resolution melting (HRM), polymerase chain reaction restriction fragment length polymorphism (PCR‐RFLP), and allelic discrimination PCR (TaqMan PCR). Discordant cases were assessed with COBAS 4800 KRAS CE‐IVD assay. Among the 674 tumor specimens, 1.5% (10/674) had excessive DNA degradation and could not be analyzed. KRAS mutations were detected in 38.0% (256/674) of the analysable specimens (82.4% in codon 12 and 17.6% in codon 13). Among 613 specimens in whom all three techniques were used, 12 (2.0%) cases of discordance between the three techniques were observed. 83.3% (10/12) of the discordances were due to PCR‐RFLP as confirmed by COBAS 4800 retrospective analysis. The three techniques were statistically comparable (κ > 0.9; P < 0.001). From these results, optimization of the routine procedure consisted of proceeding to systematic KRAS detection using HRM and TaqMan and PCR‐RFLP in case of discordance and allowed significant decrease in delays. The results showed an excellent correlation between the three techniques. Using HRM and TaqMan warrants high‐quality and rapid‐routine KRAS mutation detection in paraffin‐embedded tumor specimens. The new procedure allowed a significant decrease in delays for reporting results, enabling rational prescription of first‐line‐targeted therapy in mCRC.

Evaluation and validation of HPV real-time PCR assay for the detection of HPV DNA in oral cytobrush and FFPE samples

Specific HPV genotypes have been recognized as risk factors inducing head and neck cancers (HNC). The aim of this study was to validate a real-time PCR assay to detect accurately High Risk HPV DNA in Formalin Fixed Paraffin Embedded (FFPE) and oral cytobrush samples and compare the results with conventional PCR. Repeatability, reproducibility and limit of detection of Cobas assay were estimated for oral cytobrush and FFPE samples of patients with HNC. 53 samples of patients with a HNC were then used for assay comparison with conventional PCR. Finally, 26 samples of patients with anogenital neoplasia cancer were analyzed as control and assays comparison. Among the 53 samples of patients with HNC, 12 (26.7%) were HPV positive, 33 (73.3%) were HPV negative and 8 (15.1%) were non contributive with the Cobas assay. Among the 26 samples of patients with anogenital neoplasia, 15 (57.7%) were HPV positive and 11 were HPV negative (42.3%). One sample was found with an HPV 16 and HPV 18 co-infection. Only 3 samples were found with discrepant results. Cobas assay was found suitable for routine HPV detection with a very good repeatability and reproducibility for all HPV genotypes (CV < 0.6% and <0.4% respectively). Sensitivity and specificity for Cobas assay were 91.7% [61.5%;99.8%] and 96.9% [83.8%;99.9%] respectively. Ten nanograms of DNA were sufficient for the detection of HPV 16, HPV 18 and HPV in FFPE and oral cytobrush samples. Cobas assay was found comparable to conventional PCR and can detect accurately and rapidly HPV DNA in FFPE and oral cytobrush samples for the management of HNC and other types of HPV-associated neoplasia.

Abstract C38: Comparative evaluation of three real-time PCR assays for the detection of PIK3CA somatic mutations in formalin-fixed paraffin embedded breast carcinomas.

Background: Mutations of the PIK3CA gene encoding for the p110α catalytic subunit of Phosphatidylinositol 3-kinases (PI3K) are found in approximately 25% of breast carcinomas. Most of PIK3CA mutations are reported as activators of the PI3K/AKT/mTOR pathway and lead to resistance to anti-HER2 therapies, and have an interest in molecular diagnosis for anti-mTOR and/or anti-PI3K therapies. Highly sensitive methods are required to detect PIK3CA mutations in paraffin-embedded tumor tissues. Methods: Forty-seven formalin-fixed paraffin embedded breast carcinomas tissues samples were assessed using two allele specific assays (Cobas® PIK3CA Mutation Test and PCR ARMS Scorpions®) and one non-specific real-time high resolution melting PCR assay (HRM) after macrodissection and DNA extraction. Cobas® allow the detection of 17 mutations on exons 1, 4, 7, 9 and 20 of PIK3CA, ARMS focuses on the detection on the four common hotspots (E542K, E545K/D, H1047R and H1047L) representing 90% of PIK3CA mutations and HRM allows the detection of all mutations located on the whole exons 9 and 20 of PIK3CA. Kappa statistics were used to compare the three assays. Results: Among the 47 samples, 18 (38.3%) were found to bear a PIK3CA mutation with Cobas®, 13 (27.7%) with ARMS and 19 (40.4%) with HRM. One sample was found to bear 3 mutations of PIK3CA (E542K, H1047X and H1049R). Calculated kappa were 0.893 (p<1.10-9) between Cobas® and HRM and 0.659 (p<1.10-9) between Cobas® and ARMS. Because the ARMS assay only focused on the 4 most common PIK3CA mutations, more than 10% of the samples were identified as false negative wild-type tumors although they had a mutation of PIK3CA as identified with Cobas® and HRM assays. Conclusion: Cobas® and HRM assays allowed to detect more extensively PIK3CA mutations than ARMS assay. ARMS assay only focusing on the 4 hotspots PIK3CA mutations seems to be inadequate with routine use since generating false negative PIK3CA wild-type results. Cobas® and HRM assays are significantly comparable for the detection of PIK3CA mutations, but Cobas® enables the characterization of the mutation. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C38. Citation Format: Alexandre Harle, Maeva Lion, Marie Rouyer, Nicola Lozano, Jean-Louis Merlin. Comparative evaluation of three real-time PCR assays for the detection of PIK3CA somatic mutations in formalin-fixed paraffin embedded breast carcinomas. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C38.

Validation de méthode selon la norme ISO 15189 et le SH GTA 04 : application à la détection des mutations KRAS par méthode TaqMan

Since January 16(th) 2010, the French legislation requires that the medical laboratories must be accredited according to ISO 15189 standards. Thus, all medical laboratories in France must be accredited for at least part of their biological tests before the end of October 2013. Molecular biology tests are also concerned by the accreditation. Validation of molecular biology methods is made difficult, for reasons related to the methods, but also by the type of analytes that are basically rare. This article describes the validation of the qualitative detection of KRAS mutations in metastatic colorectal cancer using TaqMan PCR according to ISO 15189 and to the technical guide for accreditation in Human Health, SH-GTA-04, edited by the COFRAC.