Six-Color Multiplex Digital PCR Assays for Comprehensive Screening and Identification of Multiple Driver Mutations Associated with Pancreatic Carcinogenesis.

Sensitive Detection of KRAS Mutations in Archived Formalin-Fixed Paraffin-Embedded Tissue Using Mutant-Enriched PCR and Reverse-Hybridization

Molecular Analysis to Detect Pancreatic Ductal Adenocarcinoma in High-Risk Groups

Should Oncologists Be Aware in Their Clinical Practice of <i>KRAS</i> Molecular Analysis?

[Background] It is well known that emerging mutations which is not found in the primary tumor exist in metastatic tumor and molecular therapy induce emerging mutation. Liquid biopsy, which includes circulating tumor cell (CTC) and circulating tumor DNA (ctDNA), may help detecting this spatial and temporal heterogeneity. We have reported that emerging KRAS mutation can be detected by using ctDNA (Yamada et al, Cancer Science 2016). However, mutation detection by using CTC has been difficult because enough amount of DNA cannot be extracted from CTC. Currently we have been able to collect more CTC than before, by using a new device which uses 3 antibodies (EpCAM, Her2, Trop2). In this study, we evaluated the potential to detect colorectal cancer (CRC) related gene mutations from CTC, and compared it with ctDNA. [Methods] Cohort 1: This cohort included untreated CRC patients. Tumor tissue was collected from each patient, either by primary surgery or by colonoscopic biopsy. DNA was extracted from tumor tissue and was analyzed using Next Generation Sequencing (NGS). Ten mL of whole blood was also collected from the same patient. CTC, serum and white blood cell (WBC) was collected by using the CTC recovery machine (Ion Torrent Liquid Biopsy Instrument®). Cytokeratin positive, DAPI positive, CD45 negative cells were defined as CTC. DNA was extracted from each sample (CTC-DNA, ctDNA, WBC-DNA) and was analyzed using NGS. Cohort 2: This cohort included unresectable CRC patients with KRAS mutation in their primary tumor. All patients in this cohort were under treatment or after completion of chemotherapy. CTC and ctDNA was collected in the same method as cohort 1. KRAS mutations of CTC and ctDNA were detected by using digital PCR (dPCR). [Results] Cohort 1: We enrolled 16 CRC patients (stage II: n=2, stage III: n=2, stage IV: n=12). A total of 30 somatic, hotspot mutations were detected from tumor tissue DNA. The median number of the detected mutation for each patient was 2 (0-4). The most frequent gene mutation was APC, followed by KRAS and TP53. In all patients, CTC was successfully collected. The median number of the CTC was 34 cells (5-94). However, only 6 somatic, mutations were detected from CTC-DNA. Conversely, 16 somatic mutations were detected from ctDNA. Cohort 2: We enrolled 14 stage IV CRC patients with KRAS mutation in their primary tumor. CTC was collected from 9 patients but not from 5 patients. In the 9 patients CTC was collected, the median number of the collected CTC was 26 cells (5-121). By dPCR, KRAS mutation was detected in 2 patients (2/14) from CTC-DNA, and 4 patients (4/14) from ctDNA. [Conclusions] The new CTC capturing technology using 3 antibodies can improve detection rate and yield of CTC. However, in patients undergoing chemotherapy, the amount of CTC and ctDNA drastically reduces. At present, ctDNA is superior to CTC in potential to detect mutations, and dPCR is more sensitive than NGS to detect mutations. Citation Format: Kohki Takeda, Takeshi Yamada, Michihiro Koizumi, Seiichi Shinji, Yasuyuki Yokoyama, Goro Takahashi, Masahiro Hotta, Takuma Iwai, Keisuke Hara, Hiroyasu Furuki, Eiji Uchida. Detection of colorectal cancer related gene mutations from CTC and ctDNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4594.

KRAS mutations play an important role in pancreatic cancer. GNAS mutations were discovered in intraductal papillary mucinous neoplasms (IPMN).Our aim was to identify the frequency of KRAS and GNAS mutations in pancreatic cystic neoplasms and pancreatic ductal adenocarcinoma (PDAC).Sixty-eight surgically resected formalin fixed, paraffin embedded pancreatic specimens were analyzed, including: 1) benign (20 serous cystadenoma (SCA)), 2) pre-malignant (10 mucinous cystic neoplasm (MCN), 10 branch duct intraductal papillary mucinous neoplasm (BD-IPMN), 9 main duct IPMN (MD-IPMN)), 3) malignant (19 PDAC). Total nucleic acid extraction was performed. KRAS codon 12/13 and GNAS codon 201 mutations were interrogated via targeted sequencing using the Ion Torrent's Personal Genome Machine (PGM).Mean age of 68 patients was 61.9±8.4 with 72% female. KRAS and GNAS mutations were more common in PDAC and IPMN. KRAS mutations predominated in PDAC compared to pancreatic cysts (16/19, 84% versus 10/49, 20%; P&lt;0.001). GNAS mutations were more common in IPMN compared to non-IPMN lesions (8/19, 42% versus 2/49, 4%; P=0.0003). No GNAS mutations were detected in PDAC and MCN while 2 SCA carried GNAS mutations. Double mutations with KRAS and GNAS were only present in IPMN (5/19 versus 0/30 SCA and MCN, P=0.006).KRAS and GNAS mutations were more common in PDAC and IPMN with KRAS mutations primarily in PDAC and GNAS mutations more frequent in IPMN. No GNAS mutations occurred in MCN and double mutations were only present in IPMN.

Ultrasensitive Detection of KRAS2 Mutations in Bile and Serum from Patients with Biliary Tract Carcinoma Using LigAmp Technology

BackgroundThe prevalence and clinical significances of KRAS, GNAS, and RNF43 mutations in patients with pancreatic intraductal papillary mucinous neoplasm (IPMN) remain elusive. To evaluate the incidence of the gene mutations and clinicopathologic differences between KRAS and GNAS mutations in pancreatic cystic lesions, we performed a meta-analysis of published 33 KRAS, 11 GNAS, and 4 RNF43 studies including 1253, 835, and 143 cases, respectively.MethodsWe pooled the results of relevant studies identified using the PubMed and EMBASE databases. The effect sizes of outcome parameters were computed by the prevalence rate, weighted mean difference, or odds ratio (OR) using a random-effects model.ResultsThe pooled prevalence of KRAS, GNAS, and RNF43 mutations in IPMN was 61, 56, and 23 %, respectively. The KRAS (OR 7.4 and 71.2) and GNAS (OR 30.2 and 15.3) mutations were more frequently found in IPMNs than in mucinous cystic neoplasms and in serous cystadenomas, respectively. Of the microscopic subtypes of IPMN, KRAS and GNAS were frequently mutated in gastric type (OR 2.7, P < 0.001) and intestinal type (OR 3.0, P < 0.001), respectively. KRAS mutation was infrequently found in high-grade dysplasia lesions of IPMN (OR 0.6, P = 0.032). GNAS mutation was associated with male (OR 1.9, P = 0.012).ConclusionsThis meta-analysis supports that KRAS and GNAS mutations could be diagnostic markers for IPMN. In addition, the frequencies of KRAS and GNAS mutations in IPMNs are highly variable according to the microscopic duct subtypes, reflecting their independent roles in the IPMN-adenocarcinoma sequence.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-016-2847-4) contains supplementary material, which is available to authorized users.

Pancreatic cyst fluids (PCFs) enriched in tumor-derived DNA are a potential source of new biomarkers. The study aimed to analyze germinal variants and mutational profiles of cell-free (cf)DNA shed into the cavity of pancreatic cysts. The study cohort consisted of 71 patients who underwent endoscopic ultrasound fine-needle aspiration of PCF. Five malignant cysts, 19 intraductal papillary mucinous neoplasms (IPMNs), 11 mucinous cystic neoplasms (MCNs), eight serous cystic neoplasms (SCNs), and 28 pseudocysts were identified. The sequencing of 409 genes included in Comprehensive Cancer Panel was performed using Ion Proton System. The mutation rate of the KRAS and GNAS canonical loci was additionally determined using digital PCR. The number of mutations detected with NGS varied from 0 to 22 per gene, and genes with the most mutations were: TP53, KRAS, PIK3CA, GNAS, ADGRA2, and APC. The frequencies of the majority of mutations did not differ between non-malignant cystic neoplasms and pseudocysts. NGS detected KRAS mutations in malignant cysts (60%), IPMNs (32%), MCNs (64%), SCNs (13%), and pseudocysts (14%), with GNAS mutations in 20%, 26%, 27%, 13%, and 21% of samples, respectively. Digital PCR-based testing increased KRAS (68%) and GNAS (52%) mutations detection level in IPMNs, but not other cyst types. We demonstrate relatively high rates of somatic mutations of cancer-related genes, including KRAS and GNAS, in cfDNA isolated from PCFs irrespectively of the pancreatic cyst type. Further studies on molecular mechanisms of pancreatic cysts malignant transformation in relation to their mutational profiles are required.

KRAS gene mutations in codon 12 are detected remarkably in high occurrence in Iraqi patients with colorectal adenocarcinoma. This study aims to investigate the mutations in codon 12 of this gene in colorectal tumor specimens. Fresh biopsies samples were collected from one hundred and twenty colorectal cancer (CRC)patients and one hundred (ulcerative colitis) as non-malignant negative control for revealing mutations in KRAS (codon 12) using restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR), mutations were compared between patients and negative control, clinicopathologic features (age, gender, tumor size, grade, stage and tumor site) were compared between mutated and wild-type KRAS in CRC patients. Heterozygous mutations in KRAS codon 12 were significant only in individuals with CRC 52 of 120 (43.33%) when compared with non-malignant control group (ulcerative colitis) 0 (0%) (P<0.001), Odd ratio was 1.765; 95% CI was (1.765-2.202), other CRC patients were present in wild-type homozygotes 68(56.66%). High mean age, well-moderate grade, stage II and recto-sigmoid tumor was significant in association with heterozygous KRAS mutation in codon 12 than homozygous wild-type of CRC patients (P<0.05). Mutation in codon 12 KRAS is regarded as aprognostic marker used for early diagnosis of CRC, also it determines which patient responds to anti-EGFR targeted antibodies treatment.

Background: Oncogenic KRAS mutations can be used to predict a lack of response to epidermal growth factor receptor (EGFR) blockade. KRAS status is usually determined by biopsy from the primary site of colorectal cancer (CRC). However, the genomic profiles of primary tumors and metastases are not always concordant because of intrinsic molecular heterogeneity. Furthermore, chemotherapeutic agents and targeted drugs can alter the tumor molecular landscape. We exploited circulating tumor DNA (ctDNA) to genotype colorectal tumors and track clonal evolution during treatment involving EGFR blockade. To account for these spatial and temporal changes, the genomic profiles of patients with CRC can be repeatedly evaluated during the course of therapy. In this study, we evaluated the utility of KRAS mutation detection using ctDNA before and during chemotherapy. Method: Experiment 1: We enrolled 46 metastatic colorectal cancer patients. Before starting chemotherapy, ctDNA was purified from 1 mL serum using the QIAamp circulating nucleic acid kit. We detected nine KRAS (G12A, G12R, G12D, G12C, G12S, G12V, G13D, Q61H, and Q61R) mutations using the Invader method and digital PCR. Experiment 2: Fifteen patients were treated with systemic chemotherapy including EGFR blockade. ctDNA was extracted from these patients every 2 months until disease progression, and the KRAS mutation was detected in nine. Results: Experiment 1: KRAS mutations in circulating tumor DNA were detected in 88% (14/16) of patients with KRAS mutations in their primary tumor, but in 10% (10/30) of patients without KRAS mutations in their primary tumors. Experiment 2: The response rate was 87% (13/15). In two non-responders, KRAS mutations in ctDNA were detected before chemotherapy. Disease progression was identified in five patients and KRAS mutations in ctDNA were detected in all patients; the five patients with disease progression included the two non-responders in whom KRAS mutations in ctDNA were detected before chemotherapy. However, the genotypes detected after disease progression were different from those detected before chemotherapy (G13D to G12C and Q61R to Q61H). New KRAS mutations occurred in codon 12 in the other three patients in whom no KRAS mutations in ctDNA were detected before chemotherapy. New KRAS mutations in codon 61 disappeared during chemotherapy and disease progression was not detected at that time in two patients. Discussion: It has been reported in a retrospective study that new KRAS mutations were detected in patients who acquired resistance to EFGR blockade, and many of them were in codon 61. However, new KRAS mutations in codon61 can disappear during treatment involving EGFR blockade. This phenomenon may indicate that new KRAS mutations in ctDNA do not always indicate acquired resistance to chemotherapy involving EGFR blockade. Citation Format: Takeshi Yamada, Hayato Kan, Takuma Iwai, Goro Takahashi, Michihiro Koizumi, Akihisa Matsuda, Seiichi Shinji, Yasuyuki Yokoyama, Atsushi Tatsguchi, Tetsuro Kawagoe, Shiro Kitano, Masato Nakayama, Satoshi Matsumoto, Keiichiro Ohta, Eiji Uchida. Prediction of acquired resistance in colorectal cancer patients treated with EGFR blockade by detection of a new KRAS mutation in ccfDNA. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3135.

Rapid Screening Assay for KRAS Mutations by the Modified Smart Amplification Process

Identification of a Mosaic Activating Mutation in GNA11 in Atypical Sturge-Weber Syndrome

Mo1212 Correlation of GNAS Mutation With Diagnostic Information in Pancreatic Cyst Fluids

Circulating tumor cell becomes an urgent issue in personalized cancer treatment studies. AS the liquid biopsy, many previous reports showed the number and the characteristic of CTC are very important for patient prognosis. In cancer therapy, specific gene mutation in primary tumor decides the therapy strategy and drug response. However, the patient who cannot perform an operation or take biopsy would become a risk factor for treatment and affect therapy outcome. Hence, drug treatable related gene mutation detection by CTC is necessary. Platforms for mutation detection in tissue were well established but less useful for CTC. Most of them were lower efficacy for rare cell or DNA. In order to solve this dilemma, scientists use next-generation sequence (NGS) or droplet digital PCR (ddPCR) for gene mutation detection. But, in most clinical situations, we only have to follow-up some specific gene mutation, instead of many mutant types. Thus, we eager to develop a fast and low-cost methodology for drug treatable related gene mutation detection by CTC. In this study, 4mL blood was collected by EDTA vacuum blood collection tube from 37 patients. Whole blood was processed with RBC lyse steps, then added CD45 and glycophorin A antibodies conjugated with the nanoparticle to remove leukocyte and remained RBC. Further, Genomic DNA was extracted from negative selection cell mixture. An ARMS-PCR (Amplification-refractory mutation system PCR) combined with a touchdown PCR program was used for EGFR mutations identification. We enrolled 31 lung cancer patients. Nineteen patients’ CTC were matched with tissue mutation results (14 mutant and 5 wild-types), and 12 unmatched. In those 12 patients, 2 patients with mutant tissue but wild-type CTC due to their blood were drawn at disease-free status after long-term therapy, 4 patients with wild-type tissue but mutant CTC were still had tumor burden after therapy and need to more long-term follow-up. Overall, we have a mutation correlation rate of (19 + 2) / 31 = 68% between CTC and primary tumor. Furthermore, an LNA-PCR (Locked nucleic acid PCR) system was designed for colorectal cancer KRAS codon 12 and codon 13 mutations detection. In this platform, modified RNA was designed for blocked wild-type DNA amplification in PCR. We enrolled 6 patients with primary tumor mutant positive, 5 of 6 (83%) patients were also mutant positive in their CTC sample. In conclusion, an easy CTC isolation platform and PCR design could hugely help cancer patient mutation detection. Only less than 4 hours and 100 USD were needed for drug treatable gene mutation detection by CTC. Citation Format: Hung-Chih Lin, Yu-Lin Yang, Chia-Hsun Hsieh. Label-free circulating tumor cells isolation platform for lung cancer EGFR mutation identification and colorectal cancer with KRAS mutation detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3422.

PD-0025 Evaluation of Codon 12 and 13 KRAS Mutations as Biomarkers of Response to Panitumumab in Patients with Metastatic Colorectal Cancer