Digital PCR-Based Plasma Cell-Free DNA
K-Ras Mutations Analysis And Clinical Implication In Pancreatic Cancer
By GoPath Diagnostics
Authors: Nahid Forouzandeh, MD, Sachin Srinivasan, MD, Katia El Jurdi, MD, John Wang, PhD, Nathan Tofteland, MD, Wancai Yang, MD, William Salyers, MD, Jim Lu, MD, PhD
Affiliations: GoPath Laboratories LLC, Buffalo Grove, IL; Department of Internal Medicine, University of Kansas School of Medicine, Wichita, KS; University of Illinois at Chicago, Chicago, IL; GoPath Laboratories LLC, Buffalo Grove, IL
BACKGROUND: Pancreatic cancer is one of the most fatal cancers worldwide. In addition to patients presenting later in the disease, limitations in current testing modalities pose a challenge to early diagnosis and treatment. Somatic mutations of the K-Ras gene have been suggested as a key driver of pancreatic carcinogenesis and thus proposed as a biomarker for diagnosis and therapy. Majority of these studies utilize tissue-based methods for analyzing K-Ras mutations. In recent years, liquid biopsy assay, in particular, analysis of cell-free DNA (cfDNA), has emerged as a promising noninvasive diagnostic approach in oncology, with the respect to the identification of minimal residual diseases, monitoring treatment response, detection recurrence and metastasis, and identification of chemo-resistance mechanisms. The application of digital PCR based plasma cfDNA K-Ras mutation assay in pancreatic cancer has not been reported.
METHODS: We included patients who underwent evaluation of a pancreatic lesion detected by conventional radiography (CT/MRI) and confirmed by endoscopic ultrasound (EUS) and biopsy. The biopsies from pancreatic tissues were formalin-fixed and paraffin-embedded (FFPE) for pathological diagnosis, and the total DNA was extracted from the FFPE slides. In addition, matched blood from the same patient was collected at the time of biopsy, and plasma cfDNA was extracted. Both DNA quality and concentration were evaluated. 4.0 ng of DNA was used for droplet digital PCR (ddPCR) analysis. K-Ras G12D, G12V, G12R, G12C, G12A, G12S, and G13D mutations were analyzed by multiplexed assay reagents. The sensitivity cut-off of the multiplexed K-Ras G12/G13 assay was 1.0%.
RESULTS: cfDNA could be detected in the initiation of pathological diagnosis. Among the twelve patients with pancreatic lesions, eight (67%) were pancreatic ductal adenocarcinoma (PDAC) (head (4), body (2), tail (2) of the pancreas), one was acute pancreatitis, one was a tail cyst and two were unspecified non-malignant lesions. All the eight PDAC (100%) FFPE tissues exhibited K-Ras G12/13 mutations (Scores: 4.2-41.5%), while none of the non-malignant lesions (0%) demonstrated K-Ras mutations. Interestingly, K-Ras mutations were detected from seven of the eight PDAC patient’s plasma cfDNA (87.5%) (Scores: 1.2-20.7%) while the plasma cfDNA from patients with non-malignant lesions did not show any positive K-Ras mutations.
CONCLUSION: Digital PCR-based plasma cfDNA assay for K-Ras mutation is a promising tool for diagnosis of PDAC and is comparable to tissue-based assays. Larger prospective studies can substantiate this and explore their roles in the prediction and early detection of recurrence of PDAC.
Affiliations: GoPath Laboratories LLC, Buffalo Grove, IL; Department of Internal Medicine, University of Kansas School of Medicine, Wichita, KS; University of Illinois at Chicago, Chicago, IL; GoPath Laboratories LLC, Buffalo Grove, IL
BACKGROUND: Pancreatic cancer is one of the most fatal cancers worldwide. In addition to patients presenting later in the disease, limitations in current testing modalities pose a challenge to early diagnosis and treatment. Somatic mutations of the K-Ras gene have been suggested as a key driver of pancreatic carcinogenesis and thus proposed as a biomarker for diagnosis and therapy. Majority of these studies utilize tissue-based methods for analyzing K-Ras mutations. In recent years, liquid biopsy assay, in particular, analysis of cell-free DNA (cfDNA), has emerged as a promising noninvasive diagnostic approach in oncology, with the respect to the identification of minimal residual diseases, monitoring treatment response, detection recurrence and metastasis, and identification of chemo-resistance mechanisms. The application of digital PCR based plasma cfDNA K-Ras mutation assay in pancreatic cancer has not been reported.
METHODS: We included patients who underwent evaluation of a pancreatic lesion detected by conventional radiography (CT/MRI) and confirmed by endoscopic ultrasound (EUS) and biopsy. The biopsies from pancreatic tissues were formalin-fixed and paraffin-embedded (FFPE) for pathological diagnosis, and the total DNA was extracted from the FFPE slides. In addition, matched blood from the same patient was collected at the time of biopsy, and plasma cfDNA was extracted. Both DNA quality and concentration were evaluated. 4.0 ng of DNA was used for droplet digital PCR (ddPCR) analysis. K-Ras G12D, G12V, G12R, G12C, G12A, G12S, and G13D mutations were analyzed by multiplexed assay reagents. The sensitivity cut-off of the multiplexed K-Ras G12/G13 assay was 1.0%.
RESULTS: cfDNA could be detected in the initiation of pathological diagnosis. Among the twelve patients with pancreatic lesions, eight (67%) were pancreatic ductal adenocarcinoma (PDAC) (head (4), body (2), tail (2) of the pancreas), one was acute pancreatitis, one was a tail cyst and two were unspecified non-malignant lesions. All the eight PDAC (100%) FFPE tissues exhibited K-Ras G12/13 mutations (Scores: 4.2-41.5%), while none of the non-malignant lesions (0%) demonstrated K-Ras mutations. Interestingly, K-Ras mutations were detected from seven of the eight PDAC patient’s plasma cfDNA (87.5%) (Scores: 1.2-20.7%) while the plasma cfDNA from patients with non-malignant lesions did not show any positive K-Ras mutations.
CONCLUSION: Digital PCR-based plasma cfDNA assay for K-Ras mutation is a promising tool for diagnosis of PDAC and is comparable to tissue-based assays. Larger prospective studies can substantiate this and explore their roles in the prediction and early detection of recurrence of PDAC.