Mariana Petaccia de Macêdo1, Fernanda Machado de Melo2, Bianca Cristina Garcia Lisboa2, Louise D Brot Andrade3, Maria Dirlei Ferreira de Souza Begnami4, Samuel Aguiar Junior5, Heber Salvador de Castro Ribeiro6, Fernando Augusto Soares3, Dirce Maria Carraro7, Isabela Werneck da Cunha3. 1. Diagnostic Molecular Pathology Laboratory, Anatomic Pathology Department, A.C. Camargo Cancer Center, Brazil; Laboratory of Investigative Pathology, CIPE/A.C. Camargo Cancer Center, Brazil. Electronic address: maripetaccia@gmail.com. 2. Diagnostic Molecular Pathology Laboratory, Anatomic Pathology Department, A.C. Camargo Cancer Center, Brazil. 3. Diagnostic Molecular Pathology Laboratory, Anatomic Pathology Department, A.C. Camargo Cancer Center, Brazil; Laboratory of Investigative Pathology, CIPE/A.C. Camargo Cancer Center, Brazil. 4. Laboratory of Investigative Pathology, CIPE/A.C. Camargo Cancer Center, Brazil. 5. Surgical Oncology Department, A.C. Camargo Cancer Center, Brazil. 6. Abdominal Surgery Department, A.C. Camargo Cancer Center, Brazil. 7. Diagnostic Molecular Pathology Laboratory, Anatomic Pathology Department, A.C. Camargo Cancer Center, Brazil; Laboratory of Genomics and Molecular Biology, CIPE/ A.C. Camargo Cancer Center, Brazil.
Abstract
INTRODUCTION: Inhibition of EGFR is a strategy for treating metastatic colorectal cancer (CRC) patients. KRAS sequencing is mandatory for selecting wild-type tumor patients who might benefit from this treatment. DNA from formalin-fixed paraffin-embedded (FFPE) tissues is commonly used for routine clinical detection of mutations, and its amplification succeeds only when all preanalytical histological processes have been controlled. In cases that are not properly processed, the DNA results can be poor, with low peak pyrosequencing findings. We designed and tested a pair of forward and reverse primers for a nested PCR method, followed by pyrosequencing, in a single Latin American institution series of 422 unselected CRC patients, correlating KRAS mutations with pathological and clinical data. MATERIALS AND METHODS: Patient DNA samples from tumors were obtained by scraping or laser microdissection of cells from FFPE tissue and extracted using a commercial kit. DNA was first amplified by PCR using 2 primers that we designed; then, nested PCR was performed with the amplicon from the preamplification PCR using the KRAS PyroMark™ Q96 V2.0 kit (Qiagen). Pathological data were retrieved from pathology reports. RESULTS: KRAS mutation was observed in 33% of 421 cases. Codon 12 was mutated in 76% of cases versus codon 13 in 24%. Right-sided CRCs harbored more KRAS mutations than left-sided tumors, as did tumors that presented with perineural invasion. CONCLUSION: Our findings in this Latin American population are consistent with the literature regarding the frequency of KRAS mutations in CRC, their distribution between codons 12 and 13, and type of nucleotide substitution. By combining nested PCR and pyrosequencing, we achieved a high rate of conclusive results in testing KRAS mutations in CRC samples - a method that can be used as an ancillary test for failed assays by conventional PCR.
INTRODUCTION: Inhibition of EGFR is a strategy for treating metastatic colorectal cancer (CRC) patients. KRAS sequencing is mandatory for selecting wild-type tumorpatients who might benefit from this treatment. DNA from formalin-fixed paraffin-embedded (FFPE) tissues is commonly used for routine clinical detection of mutations, and its amplification succeeds only when all preanalytical histological processes have been controlled. In cases that are not properly processed, the DNA results can be poor, with low peak pyrosequencing findings. We designed and tested a pair of forward and reverse primers for a nested PCR method, followed by pyrosequencing, in a single Latin American institution series of 422 unselected CRC patients, correlating KRAS mutations with pathological and clinical data. MATERIALS AND METHODS:Patient DNA samples from tumors were obtained by scraping or laser microdissection of cells from FFPE tissue and extracted using a commercial kit. DNA was first amplified by PCR using 2 primers that we designed; then, nested PCR was performed with the amplicon from the preamplification PCR using the KRAS PyroMark™ Q96 V2.0 kit (Qiagen). Pathological data were retrieved from pathology reports. RESULTS:KRAS mutation was observed in 33% of 421 cases. Codon 12 was mutated in 76% of cases versus codon 13 in 24%. Right-sided CRCs harbored more KRAS mutations than left-sided tumors, as did tumors that presented with perineural invasion. CONCLUSION: Our findings in this Latin American population are consistent with the literature regarding the frequency of KRAS mutations in CRC, their distribution between codons 12 and 13, and type of nucleotide substitution. By combining nested PCR and pyrosequencing, we achieved a high rate of conclusive results in testing KRAS mutations in CRC samples - a method that can be used as an ancillary test for failed assays by conventional PCR.
Authors: Humaid O Al-Shamsi; Jeremy Jones; Yazan Fahmawi; Ibrahim Dahbour; Aziz Tabash; Reham Abdel-Wahab; Ahmed O S Abousamra; Kenna R Shaw; Lianchun Xiao; Manal M Hassan; Benjamin R Kipp; Scott Kopetz; Amr S Soliman; Robert R McWilliams; Robert A Wolff Journal: J Gastrointest Oncol Date: 2016-12
Authors: Mariana Petaccia de Macedo; Fernanda M Melo; Heber Salvador C Ribeiro; Marcio C Marques; Luciane T Kagohara; Maria Dirlei Begnami; Julio C Neto; Júlia S Ribeiro; Fernando A Soares; Dirce M Carraro; Isabela W Cunha Journal: Am J Cancer Res Date: 2017-09-01 Impact factor: 6.166
Authors: Monica Molano; Sepehr N Tabrizi; Suzanne M Garland; Jennifer M Roberts; Dorothy A Machalek; Samuel Phillips; David Chandler; Richard J Hillman; Andrew E Grulich; Fengyi Jin; I Mary Poynten; David J Templeton; Alyssa M Cornall Journal: PLoS One Date: 2016-08-16 Impact factor: 3.240