Colorectal cancer (CRC) is one of the most common cancers worldwide. In the United States is currently the third deadliest cancer with more than 1 million patients diagnosed annually, of which 50% will develop metastatic disease [1]. In some subtypes of CRC, the Kras mutation status has emerged as an important predictive marker for the response to treatment with anti-EGFR drugs in patients with metastatic CRC [2, 3]. Several molecular techniques characterized by different sensitivities, specificities and complexities are currently used in research and clinical studies for the detection of KRAS mutations [4]. However, there are still several difficulties when analysing KRAS mutations using the existing assays, particularly with regard to low sensitivity, it's time-consuming, and the need for large instruments. Here, we explain the development of a new method to detect simultaneously the main KRAS mutations on a portable Q3 Lab-Disk platform (STMicroelectronics) based on Real-Time PCR methodology (Figure 1). Material and methods: The developed test for detection of KRAS includes the eight common hotspot spanning in codons 12 and 13 of exon 2 (Table 1). The KRAS point mutation panel was identified by the use of public database annotations including the most commonly observed single nucleotide variations/polymorphisms (SNPs). The selection criteria were based on population frequency data and their involvement to CRC cancer. Specificity assay: Genotyping of human KRAS gene was performed by Q3 Lab-Disk platform developed by STMicroelectronics for efficient nucleic acids detection [5]. Briefly, TaqMan Genotyping assay was performed using wild-type and mutant allele specific primers pairs (table 1) in order to confirm the mutational status of KRAS gene in approximatively 10 ng of Human genomic DNA. The Cycle Threshold (Ct) values of the PCR were compared with those obtained with the standard equipment Light-Cycler 1.5 (Roche Diagnostic, USA) in order to determine the performance of the new Q3 Lab-Disk platform. Results: The Q3 Lab-Disk platform was able to identify the mutations in KRAS that are predictive of prognosis of CRC and individual response to therapy. The performance was comparable in terms of threshold cycles (Cts) and amplification plots to those of other commercial instruments (Figure 2). Discussion: Q3 Lab-Disk platform is an innovative platform able to detect point mutations in Human Genome whit high specificity and sensibility. The technological innovation achieved makes the Q3 Lab-Disk platform competitive in the human genes genotyping with a considerable advantage over traditional platforms, which results in a reduction in costs and time required for the analysis, maintaining high sensitivity. These features make the Q3 LabDisk platform a reliable Point-of-Care for rapid and accurate genotyping of human DNA samples applicable to various medical and biological porpoise.
KRAS MUTATION TESTING ON MINIATURIZED Q3 LAB-DISK DEVICE
Rosario Iemmolo;Maria Guarnaccia;Sebastiano Cavallaro;
2018
Abstract
Colorectal cancer (CRC) is one of the most common cancers worldwide. In the United States is currently the third deadliest cancer with more than 1 million patients diagnosed annually, of which 50% will develop metastatic disease [1]. In some subtypes of CRC, the Kras mutation status has emerged as an important predictive marker for the response to treatment with anti-EGFR drugs in patients with metastatic CRC [2, 3]. Several molecular techniques characterized by different sensitivities, specificities and complexities are currently used in research and clinical studies for the detection of KRAS mutations [4]. However, there are still several difficulties when analysing KRAS mutations using the existing assays, particularly with regard to low sensitivity, it's time-consuming, and the need for large instruments. Here, we explain the development of a new method to detect simultaneously the main KRAS mutations on a portable Q3 Lab-Disk platform (STMicroelectronics) based on Real-Time PCR methodology (Figure 1). Material and methods: The developed test for detection of KRAS includes the eight common hotspot spanning in codons 12 and 13 of exon 2 (Table 1). The KRAS point mutation panel was identified by the use of public database annotations including the most commonly observed single nucleotide variations/polymorphisms (SNPs). The selection criteria were based on population frequency data and their involvement to CRC cancer. Specificity assay: Genotyping of human KRAS gene was performed by Q3 Lab-Disk platform developed by STMicroelectronics for efficient nucleic acids detection [5]. Briefly, TaqMan Genotyping assay was performed using wild-type and mutant allele specific primers pairs (table 1) in order to confirm the mutational status of KRAS gene in approximatively 10 ng of Human genomic DNA. The Cycle Threshold (Ct) values of the PCR were compared with those obtained with the standard equipment Light-Cycler 1.5 (Roche Diagnostic, USA) in order to determine the performance of the new Q3 Lab-Disk platform. Results: The Q3 Lab-Disk platform was able to identify the mutations in KRAS that are predictive of prognosis of CRC and individual response to therapy. The performance was comparable in terms of threshold cycles (Cts) and amplification plots to those of other commercial instruments (Figure 2). Discussion: Q3 Lab-Disk platform is an innovative platform able to detect point mutations in Human Genome whit high specificity and sensibility. The technological innovation achieved makes the Q3 Lab-Disk platform competitive in the human genes genotyping with a considerable advantage over traditional platforms, which results in a reduction in costs and time required for the analysis, maintaining high sensitivity. These features make the Q3 LabDisk platform a reliable Point-of-Care for rapid and accurate genotyping of human DNA samples applicable to various medical and biological porpoise.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
