br genetic testing was performed
15) genetic testing was performed (Figure 1). Clinical parameters, including age, gender, histologic diagnosis, Eastern Cooperative Oncology Group performance status, and tumor anatomic location at initial presentation were obtained by review of the medical re-cords. The tumor sites were classified as right-sided colon (ileocecal junction, cecum, ascending colon, hepatic flexure, and transverse colon), left-sided colon (splenic flexure, descending colon, and sigmoid colon), or rectum. All tumors were staged according to the TNM staging system of the American Joint Committee on Cancer (7th version, 2009).
HER2 IHC and FISH
HER2 status of tumor tissue was determined with formalin-fixed and paraffin-embedded (FFPE) samples using routine IHC and FISH methods. According to the American Society of Clinical Oncologists/College of American Pathologists guideline recommendations,16 IHC scores of 0 and 1þ were considered HER2-negative (HER2 ), whereas an IHC score of 3þ was defined as HER2þ. An IHC score of 2þ was considered
Figure 1 Flow Chart for Measurement of HER2 Expression and RAS/BRAF Genetic Analysis
Abbreviations: ctDNA ¼ circulating tumor DNA; FISH ¼ fluorescence in situ hybridization; HER2 ¼ human epidermal growth factor Pronase E 2.
Qing Wei et al
Figure 2 Fluorescence in Situ Hybridization (FISH) of Human Epidermal Growth Factor Receptor 2 (HER2) in 2 Patients. Green Signals Represent the Centromeric Region of Chromosome 17, Red Signals Represent the HER2 Gene on Chromosome 17. A, FISH-positive, FISH:HER2/Chr17 [ 7.5; B, FISH-positive, FISH:HER2/Chr17 [ 3.8
equivocal and the sample was subjected to further testing by FISH; a tumor was considered to be HER2þ if it had an IHC score of 2þ plus FISH results showing a threshold ratio > 2.0 between the HER2 CN and chromosome 17 centromere (CEP17). Figure 2 shows HER2 amplification by FISH in 2 patients with metastatic CRC.
KRAS, NRAS, and BRAF Status
All patients were evaluated for KRAS, NRAS, and BRAF muta-tions by Sanger sequencing. Genomic DNA was extracted from FFPE sections with 50% tumor cells (sections with tumor cell content lower than 50% were microdissected) using E.Z.N.A.FFPE DNA Kit (Lot. D3399-01, OMEGA) according to the
manufacturer’s instructions. All genomic DNA samples were stored at 20 C until further analysis. DNA fragments including the KRAS/NRAS gene (exon 2/3/4) and exon 15 of the BRAF gene were amplified by polymerase chain reaction (PCR). PCR primers and amplified fragments used for RAS/BRAF testing are detailed in Table 1. Each PCR reaction consisted of 2 mL 10 LA PCR buffer II, 2 mL 2.5 mmol/L dNTPs, 0.1 mL LA Taq (DRR200A, TAKARA), 2 mL genomic DNA, 0.5 mL 10 mmol/L forward primer, and 0.5 mL 10 mmol/L reverse primer in a final volume of 20 mL. The cycling conditions were 95 C for 5 minutes, 45 cycles of 95 C for 30 seconds, 56 C for 45 seconds, and 72 C for 20 seconds, and final extension at 72 C for 5 minutes. The detailed sequencing procedures have been reported.17
Table 1 PCR Primers and Amplified Fragments Used for RAS/BRAF Testing
Primer Fragments, bp