KRAS Mutations In ctDNA Research Articles

Evaluation of the diagnostic and prognostic clinical values of circulating tumor DNA and cell-free DNA in pancreatic malignancies: a comprehensive meta-analysis.

The diagnostic and prognostic clinical value of circulating tumor DNA (ctDNA) and cell-free DNA (cfDNA) in pancreatic malignancies are unclear. Herein, we aimed to perform a meta-analysis to evaluate ctDNA and cfDNA as potential diagnostic and prognostic biomarkers. PRISMA reporting guidelines were followed closely for conducting the current meta-analysis. The PubMed/Medline, Scopus, and Web of Science (WoS) databases were scanned in detail to identify eligible papers for the study. A quality assessment was performed in accordance with the REMARK criteria. The risk ratios (RRs) of the diagnostic accuracy of ctDNA compared to that of carbohydrate antigen 19.9 (CA 19.9) in all disease stages and the hazard ratios (HRs) of the prognostic role of ctDNA in overall survival (OS) were calculated with 95% confidence intervals (CIs). A total of 18 papers were evaluated to assess the diagnostic accuracy and prognostic value of biomarkers related to pancreatic malignancies. The pooled analysis indicated that CA19.9 provides greater diagnostic accuracy across all disease stages than ctDNA or cfDNA (RR = 0.64, 95% CI: 0.50-0.82, p < 0.001). Additionally, in a secondary analysis focusing on prognosis, patients who were ctDNA-positive were found to have significantly worse OS (HR = 2.00, 95% CI: 1.51-2.66, p < 0.001). The findings of this meta-analysis demonstrated that CA19-9 still has greater diagnostic accuracy across all disease stages than KRAS mutations in ctDNA or cfDNA. Nonetheless, the presence of detectable levels of ctDNA was associated with worse patient outcomes regarding OS. There is a growing need for further research on this topic. https://doi.org/10.37766/inplasy2023.12.0092, identifier INPLASY2023120092.

Evaluation of the Prognostic Value of Low-Frequency KRAS Mutation Detection in Circulating Tumor DNA of Patients with Metastatic Colorectal Cancer.

KRAS mutation in tumor tissue is a well-known predictor of resistance to the treatment of anti-EGFR antibodies in metastatic colorectal cancers (mCRC). However, the prognostic value of low-frequency plasma circulating tumor DNA (ctDNA) KRAS mutation in predicting treatment resistance in pretreated mCRC patients remains controversial. This study retrospectively reviewed the clinical course, including response to anti-EGFR and anti-VEGF therapies, and changes in serum tumor marker levels along with image studies in mCRC patients with <1.5% KRAS mutations detected in plasma ctDNA by next-generation sequencing (NGS) at a single center in Taiwan. We identified six pretreated mCRC patients with low-frequency KRAS G12V/G12D/G12S/G13D mutations (variant allele frequency 0.26~1.23%) in plasma ctDNA. Co-occurring low-frequency ctDNA mutations in APC, TP53, MAP2K1, KEAP1, or CTNNB1 were also detected. Although all six patients had treatment adjustments within one month after the ctDNA genetic test, image-evident tumor progression was noted in all patients within a median of 4 months afterwards. Re-challenge therapy with a combination of anti-EGFR, anti-VEGF, and FOLFIRI chemotherapy was found to be ineffective in a patient with 0.38% KRAS G12D mutation in baseline ctDNA. Our study suggests that the detection of low-frequency KRAS mutations in ctDNA could be used as a predictor of treatment response in mCRC patients.

Abstract 5577: Detection of RAS mutations in colorectal cancer patients using DNA from extracellular vesicles

Abstract Introduction: Precision medicine is becoming increasingly important in cancer treatment, and liquid biopsy (LB) is a core technique. Circulating tumor DNA (ctDNA) is the main target for LB; however, in some patients, its sensitivity is inadequate. Detection of post-operative ctDNA, called Minimal Residual Disease (MRD), indicates cancer recurrence; however, some patients experience recurrent disease without being MRD-positive. To improve sensitivity of ctDNA detection, we focused on exosomes, small vesicles of endosomal origin that contain DNA (exoDNA) derived from cells, including cancer cells. In this study, we assayed KRAS mutations in patients with colorectal cancer using exoDNA, and analyzed the utility of biomarkers in prognosis, using a machine learning algorithm. Materials and Methods: Patients who underwent curative surgery for colorectal cancer from November 2018 to December 2020 were recruited prospectively. Blood samples were obtained before surgery, and ctDNA was extracted from 1 mL of plasma. Exosomes were isolated from this plasma, followed by exoDNA extraction. KRAS mutations in ctDNA and exoDNA were analyzed using droplet digital PCR. The Random Survival Forest (RSF) algorithm was used for variable importance analysis. Results: 300 patients (123 with KRAS mutation and 177 without) were included. The median patient age was 71 years (range: 35 - 91). Concentrations of exoDNA were significantly higher in Stage IV patients compared to patients at other stages (p &amp;lt; 0.01). Numbers of exosomes did not differ by stage. KRAS mutations were detected in exoDNA of 56 patients (45.5%) and in ctDNA of 28 patients with KRAS mutation in their tumors (22.7%, p&amp;lt;0.001). Sensitivity of exoDNA was higher than that of ctDNA in Stage II/III patients with KRAS mutations (Stage II; 33.3% vs 14.2%, p=0.02, Stage III 46.7% vs 13.3%, p=0.001). However, sensitivity of cfDNA was almost equal to that of exoDNA in stage IV patients (53.8% vs 50.0%, p = 0.78). Surprisingly, in patients without KRAS mutations in their tumors, KRAS mutations were detected in exoDNA of 24 patients (13.5%) and in ctDNA of one patient (0.5%). Of 103 stage III patients, 31 (30.1%) experienced recurrence. The RSF algorithm showed that the exoDNA concentration is the most important risk factor for recurrence, followed by a lack of adjuvant chemotherapy. Conclusion: Sensitivity of KRAS mutation detection using exoDNA is excellent. Additionally, the concentration of exoDNA appears to be a superior predictor for recurrence of Stage II/III colorectal cancer after curative surgery. Citation Format: Sho Kuriyama, Takeshi Yamada, Hiromichi Sonoda, Seiichi Shinji, Akihisa Matsuda, Kazuhide Yonaga, Takuma Iwai, Kohki Takeda, Koji Ueda, Toshimitsu Miyasaka, Shintaro Kanaka, Hiroshi Yoshida. Detection of RAS mutations in colorectal cancer patients using DNA from extracellular vesicles. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5577.

Rapid Multiplex Strip Test for the Detection of Circulating Tumor DNA Mutations for Liquid Biopsy Applications.

In the era of personalized medicine, molecular profiling of patient tumors has become the standard practice, especially for patients with advanced disease. Activating point mutations of the KRAS proto-oncogene are clinically relevant for many types of cancer, including colorectal cancer (CRC). While several approaches have been developed for tumor genotyping, liquid biopsy has been gaining much attention in the clinical setting. Analysis of circulating tumor DNA for genetic alterations has been challenging, and many methodologies with both advantages and disadvantages have been developed. We here developed a gold nanoparticle-based rapid strip test that has been applied for the first time for the multiplex detection of KRAS mutations in circulating tumor DNA (ctDNA) of CRC patients. The method involved ctDNA isolation, PCR-amplification of the KRAS gene, multiplex primer extension (PEXT) reaction, and detection with a multiplex strip test. We have optimized the efficiency and specificity of the multiplex strip test in synthetic DNA targets, in colorectal cancer cell lines, in tissue samples, and in blood-derived ctDNA from patients with advanced colorectal cancer. The proposed strip test achieved rapid and easy multiplex detection (normal allele and three major single-point mutations) of the clinically relevant KRAS mutations in ctDNA in blood samples of CRC patients with high specificity and repeatability. This multiplex strip test represents a minimally invasive, rapid, low-cost, and promising diagnostic tool for the detection of clinically relevant mutations in cancer patients.

Plasmatic KRAS Kinetics for the Prediction of Treatment Response and Progression in Patients With KRAS-mutant Lung Adenocarcinoma

IntroductionKRAS is the most common driver mutation in lung cancer. ctDNA-based assessment offers advantages over tumor as a minimally invasive method able to capture tumor heterogeneity. Monitoring KRAS mutational load in ctDNA may be useful in the management of the patients. MethodsConsecutive patients diagnosed with KRAS mutant lung adenocarcinoma in the tumor biopsy were included in this study. Plasma samples were obtained at different time points during the course of the disease. KRAS mutations in plasma were quantified using digital PCR and correlated with mutations in tumor and with radiological response and progression. ResultsTwo hundred and forty-five plasma samples from 56 patients were analyzed. The rate of detection of KRAS mutations in plasma in our previously characterized KRAS-mutant cases was 82% overall, reaching 96% in cases with more than 1 metastatic location. The dynamics of KRAS mutational load predicted response in 93% and progression in 63% of cases, 33 and 50 days respectively in advance of radiological evaluation. Progression-free survival for patients in whom ctDNA was not detectable in plasma after treatment initiation was significantly longer than for those in whom ctDNA remained detectable (7.7 versus 3.2 months; HR: 0.44, p=0.004). ConclusionsThe detection of KRAS mutations in ctDNA showed a good correlation with that in tumor biopsy and, in most cases, predicted tumor response and progression to chemotherapy in advance of radiographic evaluation. The liquid biopsies for ctDNA-based molecular analyses are a reliable tool for KRAS testing in clinical practice.

Prognostic value of absolute quantification of mutated KRAS in circulating tumour DNA in lung adenocarcinoma patients prior to therapy.

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive and accurate method for quantification of nucleic acid sequences. We used absolute quantification of mutated v‐Ki‐ras2 Kirsten rat sarcoma viral oncogene homology gene (KRAS) by ddPCR to investigate the prognostic role of mutated KRAS in patients with KRAS‐mutated lung adenocarcinomas. Pre‐treatment plasma samples from 60 patients with stages I–IV KRAS‐mutated lung adenocarcinomas were analysed for KRAS mutations. The associations between survival, detectable KRAS mutations in plasma, and the plasma concentration of mutated KRAS were assessed. Overall, 23 of 60 (38%) patients had detectable KRAS mutation in plasma. The percentage of patients with detectable mutation was 8% in stage I, 30% in stage II, 71% in stage III, and 73% in stage IV. Estimated overall median progression‐free survival (PFS) and overall survival (OS) were 26.2 months [95% confidence interval (CI) 12.5–39.9] and 50.8 months (95% CI 0–107.3), respectively. Patients with detectable mutations in plasma had significantly worse median PFS compared to patients with undetectable mutation (13.1 versus 70.1 months) and shorter median OS (20.7 versus not reached). High circulating tumour DNA (ctDNA) concentrations of mutated KRAS were significantly associated with shorter PFS [hazard ratio (HR) 1.008, 95% CI 1.004–1.012] and OS (HR 1.007, 95% CI 1.003–1.011). All associations remained statistically significant in multivariable analyses. In conclusion, ddPCR is an accurate and easily feasible technique for quantification of KRAS mutations in ctDNA. The presence of detectable KRAS mutation in plasma at baseline was associated with worse PFS and OS. High concentration of mutated KRAS in ctDNA was an independent negative prognostic factor for both PFS and OS.

Combined assay of Circulating Tumor DNA and Protein Biomarkers for early noninvasive detection and prognosis of Non-Small Cell Lung Cancer.

Purpose: Early diagnosis of lung cancer is critical to curtailing cancer-related deaths. We aimed to develop a highly sensitive assay for the analysis of circulating tumor DNA (ctDNA) to detect non-small cell lung cancer (NSCLC) in the early stages.Materials and Methods: We detected EGFR and KRAS mutations in paired plasma and tumor tissue samples from 147 NSCLC patients. Of these, EGFR/KRAS ctDNA mutations and protein biomarkers were comparatively analyzed in 87 individuals. In addition, tissue samples of 20 patients were subjected to repeat multi-gene detection, and pre- and post-operative paired samples of 28 patients were subjected to multi-gene detection. Clinical information was obtained to complement the prognostic value of the combined assay results and post-operative new ctDNA mutation status.Results: EGFR/KRAS mutations were highly consistent in ctDNA and tumor DNA. Combining the detection of EGFR and KRAS mutations in ctDNA with the detection of protein biomarkers increased cancer detection sensitivity to 74.7% (65/87). None of the healthy controls tested positive using the combined assay (100% specificity). Combined assay results independently associated with recurrence-free survival. Post-operative new ctDNA mutation status independently associated with overall survival and recurrence-free survival.Conclusion: The detection of ctDNA may be exploited for early diagnosis of NSCLC, as highlighted by the developed assay. Further, the combined assay results and post-operative new ctDNA mutation status are promising prognostic indicators in NSCLC patients.

335P Resistance to CDK4/6 inhibitors: Clinical practice use of liquid biopsy to identify KRAS-mutations in ctDNA and overexpression of CDK9 in plasma derived exosomes

Despite therapeutic improvements, all patients (pts) sooner or later acquire resistance to CDK4/6 inhibitors. Mutations in KRAS have been thought to be the main cause of resistance in several cancers, but have not been analysed extensively in breast cancer in the era of Palbociclib+Fulvestrant (P+F). In our study, using liquid biopsy, we correlated the emerging KRAS-mutated ctDNA and overexpression of CDK9 with resistance to P+F as first line metastatic treatment in HR+/HER2-Metastatic Breast Cancer (MBC).

Plasmatic KRAS Kinetics for the Prediction of Treatment Response and Progression in Patients With KRAS-mutant Lung Adenocarcinoma

IntroductionKRAS is the most common driver mutation in lung cancer. ctDNA-based assessment offers advantages over tumor as a minimally invasive method able to capture tumor heterogeneity. Monitoring KRAS mutational load in ctDNA may be useful in the management of the patients. MethodsConsecutive patients diagnosed with KRAS mutant lung adenocarcinoma in the tumor biopsy were included in this study. Plasma samples were obtained at different time points during the course of the disease. KRAS mutations in plasma were quantified using digital PCR and correlated with mutations in tumor and with radiological response and progression. ResultsTwo hundred and forty-five plasma samples from 56 patients were analyzed. The rate of detection of KRAS mutations in plasma in our previously characterized KRAS-mutant cases was 82% overall, reaching 96% in cases with more than 1 metastatic location. The dynamics of KRAS mutational load predicted response in 93% and progression in 63% of cases, 33 and 50 days respectively in advance of radiological evaluation. Progression-free survival for patients in whom ctDNA was not detectable in plasma after treatment initiation was significantly longer than for those in whom ctDNA remained detectable (7.7 versus 3.2 months; HR: 0.44, p=0.004). ConclusionsThe detection of KRAS mutations in ctDNA showed a good correlation with that in tumor biopsy and, in most cases, predicted tumor response and progression to chemotherapy in advance of radiographic evaluation. The liquid biopsies for ctDNA-based molecular analyses are a reliable tool for KRAS testing in clinical practice.

Plasma vs. serum in circulating tumor DNA measurement: characterization by DNA fragment sizing and digital droplet polymerase chain reaction.

Background Choosing the specimen type is the first step of the pre-analytical process. Previous reports suggested plasma as the optimal specimen for circulating tumor DNA (ctDNA) analysis. However, head-to-head comparisons between plasma and serum using platforms with high analytical sensitivity, such as droplet digital polymerase chain reaction (ddPCR), are limited, and several recent studies have supported the clinical utility of serum-derived ctDNA. This study aimed to compare the DNA profiles isolated from plasma and serum, characterize the effects of the differences between specimens on ctDNA measurement, and determine the major contributors to these differences. Methods We isolated cell-free DNA (cfDNA) from 119 matched plasma/serum samples from cancer patients and analyzed the cfDNA profiles by DNA fragment sizing. We then assessed KRAS mutations in ctDNA from matched plasma/serum using ddPCR. Results The amount of large DNA fragments was increased in serum, whereas that of cfDNA fragments (<800 bp) was similar in both specimens. ctDNA was less frequently detected in serum, and the KRAS-mutated fraction in serum was significantly lower than that in plasma. The differences in ctDNA fractions between the two specimen types correlated well with the amount of large DNA fragments and white blood cell and neutrophil counts. Conclusions Our results provided detailed insights into the differences between plasma and serum using DNA fragment sizing and ddPCR, potentially contributing to ctDNA analysis standardization. Our study also suggested that using plasma minimizes the dilution of tumor-derived DNA and optimizes the sensitivity of ctDNA analysis. So, plasma should be the preferred specimen type.

Enrichment technique to allow early detection and monitor emergence of KRAS mutation in response to treatment

Sensitivity of cell-free circulating tumour DNA (ctDNA) assays is often hampered by the limited quantity of intact mutant nucleotide fragments. To overcome the issue of substrate limitation in clinical applications, we developed an enrichment method utilizing pyrrole-imidazole (PI) polyamides and their ability to bind the minor groove of B-DNA. We present here a proof-of-concept experiment to enrich specific mutant KRAS alleles with biotinylated PI polyamides. We investigated the clinical feasibility of incorporating PI polyamides to detect KRAS mutations in ctDNA from 40 colorectal cancer (CRC) patients, of whom 17 carried mutations in KRAS. After enriching ctDNA with those polyamides, we used digital PCR to detect several common KRAS codon 12 mutations. Enrichment by biotinylated PI polyamides improved the sensitivity of ctDNA analysis (88.9% vs. 11.1%, P < 0.01) in 9 non-metastatic mutation-positive patients. We observed no differences in performance for the 8 metastatic subjects (100% vs. 75%, P = 0.47). In the remaining 23/40 patients with wild type KRAS codon 12, no mutant alleles were detected with or without polyamide-facilitated enrichment. Enriching B-form of ctDNA with PI polyamides significantly improved the assay sensitivity in detecting KRAS mutations in non-metastatic CRC patient samples.

Circulating Tumor DNA Analysis: Clinical Implications for Colorectal Cancer Patients. A Systematic Review.

BackgroundLiquid biopsies could improve diagnosis, prognostication, and monitoring of colorectal cancer (CRC). Mutation, chromosomal copy number alteration, and methylation analysis in circulating tumor DNA (ctDNA) from plasma or serum has gained great interest. However, the literature is inconsistent on preferred candidate markers, hampering a clear direction for further studies and clinical translation. This review assessed the potential of ctDNA analysis for clinical utility.MethodsA systematic review according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines was conducted up to December 3, 2018, followed by methodological quality assessment. Primary endpoints were accuracy for detection, prognostication, and monitoring.ResultsEighty-four studies were included. For CRC detection, sensitivity was 75% using ctDNA mutation analysis and up to 96% using copy number analysis. Septin 9 (SEPT9) hypermethylation analysis showed sensitivities of 100% and specificities of 97%. Regarding prognostication, ctDNA KRAS mutations were associated with oncological outcome and could predict response to anti–epidermal growth factor receptor therapy. For monitoring, sequential ctDNA KRAS mutation analysis showed promise for detection of relapses or therapy resistance.ConclusionsThis comprehensive overview of ctDNA candidate markers demonstrates SEPT9 methylation analysis to be promising for CRC detection, and KRAS mutation analysis could assist in prognostication and monitoring. Prospective evaluation of marker panels in clinical decision making should bring ctDNA analysis into practice.

RAS/BRAF Circulating Tumor DNA Mutations as a Predictor of Response to First-Line Chemotherapy in Metastatic Colorectal Cancer Patients.

Background Since circulating tumor DNA (ctDNA) offers clear advantages as a minimally invasive method for tumor monitoring compared with tumor tissue, we aimed to evaluate genotyping ctDNA using a next-generation sequencing- (NGS-) based panel to identify the prognostic value of mutation status in metastatic colorectal cancer (mCRC) patients with primary tumor resected and with subsequent lines of treatment in this study. Methods 76 mCRC patients treated in Beijing Chao-Yang Hospital from 2011 to 2017 were enrolled. Genotyping of RAS/BRAF in tumor tissue and ctDNA was determined by ARMS PCR and with a 40-gene panel using NGS, respectively. Patient clinicopathologic features and RAS/BRAF gene mutation status were evaluated by survival analysis for disease-free survival (DFS) and progression-free survival (PFS). Results Among 76 patients, KRAS distributions were not significantly correlated with any clinicopathologic features. The concordance between tumor tissue and ctDNA KRAS mutation was 81.25%. Mutations of RAS/BRAF had no significant impact on DFS after surgery (hazard ratio (HR), 1.205; 95% CI, 0.618 to 2.349; P = 0.5837) but prognosticated poorer PFS in subsequent first-line therapy (HR, 3.351; 95% CI, 1.172 to 9.576; P = 0.024). Conclusion ctDNA was comparable with tumor tissue for mutation detection. RAS/BRAF mutations detected in ctDNA predict a worse PFS in mCRC patients with first-line chemotherapy. Our results provide support for the prognostic value of RAS/BRAF ctDNA mutation detection in mCRC patients.

Prognostic value of EGFR and KRAS in circulating tumor DNA in patients with advanced non-small cell lung cancer: a systematic review and meta-analysis.

EGFR (exon 19 and exon 21) mutations in patients with advanced non-small cell lung cancer (NSCLC) treated by EGFR-TKIs are associated with a better survival; while KRAS mutations predict a worse prognosis. However, there are divergent findings regarding the prognostic value of EGFR and KRAS mutations in circulating tumor DNA (ctDNA). We aimed to summarize the evidence for the use of circulating EGFR and KRAS mutations as prognostic factors in advanced NSCLC patients.We searched the network databases for studies reporting progression-free survival (PFS) and overall survival (OS) stratified by EGFR or KRAS mutations in ctDNA in advanced NSCLC patients. Thirteen studies enrolling 2,293 patients were reviewed. Correlation of circulating EGFR or KRAS mutations with patients’ prognosis was assessed by meta-analysis.The pooled analyses showed that EGFR mutations in ctDNA significantly prolong PFS (HR=0.64,95% CI 0.51-0.81, I2=0%, p=0.0002), namely, in patients treated by EGFR-TKIs. There is a trend to have a prolonged OS for advanced NSCLC patients with circulating EGFR mutations who were treated by EGFR-TKIs (HR=0.79, 95% CI 0.52-1.21, I2=0, p=0.28). KRAS mutations detected in ctDNA predict a worse PFS (HR=1.83, 95% CI 1.40-2.40, p<0.0001) and OS (HR=2.07, 95% CI 1.54-2.78, p<0.00001) in advanced NSCLC patients treated by chemotherapy. Sensitivity analyses and subgroup analyses demonstrated the stability of our conclusion.Our analysis showed that EGFR mutations in ctDNA predicted a better PFS, in particular in advanced NSCLC patients treated by EGFR-TKIs. KRAS mutations in ctDNA indicated a worse PFS and OS in patients treated by chemotherapy.

Abstract 3135: Prediction of acquired resistance in colorectal cancer patients treated with EGFR blockade by detection of a new KRAS mutation in ccfDNA

Abstract 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.

Abstract B4: Use of urinary circulating tumor DNA (ctDNA) KRAS for monitoring treatment response in patients with metastatic colorectal cancer (mCRC)

Abstract Background: Radiographic imaging in patients with mCRC is the current standard of care for monitoring responses. At least 40% of patients with mCRC have tumor associated KRAS mutations, and these may be detected in tumor DNA in plasma and urine. The aim of this study was to correlate the dynamics of KRAS mutational load in urinary and plasma ctDNA with clinical responses in patients with mCRC. Methods: In a blinded biomarker study of 30 metastatic patients CRC receiving chemotherapy or surgical treatment for resectable liver metastases, interim analysis was conducted in 13 patients (7 harboring KRAS G12/13 mutation in the tumor tissue, 6 wild-type for KRAS by tissue). For KRAS-positive patients, urine and plasma specimens were obtained pre-treatment, 2 weeks after initiation of chemotherapy, and subsequently at monthly intervals. Urine ctDNA was extracted using a method that preferentially isolates short fragmented ctDNA. Quantitative analysis of KRAS G12/13 mutation load in both urine and plasma was performed with the same validated KRAS G12/13 assay, which utilizes a wild-type blocker PCR enrichment followed by a next generation sequencing-based detection (MiSeq) with standardized reporting of mutant copies per 10^5 genome equivalents. Results: In an interim analysis, patients enrolled had pre-treatment urine and plasma specimens. In addition, a subset of patients (6) had longitudinal collections for up to 17 months. Eleven of 13 patients had liver-dominant metastatic disease. All KRAS tissue positive patients were receiving FOLFOX; all KRAS tissue-negative patients were receiving FOLFIRI plus cetuximab. In all KRAS G12/13 tissue positive patients with liver metastatic disease, a urinary ctDNA KRAS mutation concordant with tissue was identified; in 2 patients with non-liver metastatic disease, KRAS mutation was discordant or unidentifiable suggesting different biology in these patients. Plasma ctDNA KRAS mutation concordant with tissue was identified in 6 of 7 patient. Overall, the dynamics of ctDNA KRAS mutation burden in urine showed stronger concordance with clinical course and lack of temporal fluctuations as compared to the dynamics of ctDNA KRAS in plasma. A significant decrease in urinary ctDNA KRAS signal was observed as early as 2 weeks on chemotherapy, and this molecular response correlated with subsequently documented radiographic response. In 1 patient with documented progressive disease, an increase in urinary ctDNA KRAS G12/13 signal was observed 2 months prior to radiographic progression. Finally, 5 of 6 patients who tested negative for KRAS G12/13 mutations by tumor biopsy also tested negative in urine and plasma; 1 additional tissue KRAS wild-type patient tested positive for KRAS G12D mutation by urine and plasma ctDNA. Conclusions: In a proof of concept study, urine ctDNA was shown to carry a concordant KRAS mutation with tissue of mCRC patients. ctDNA analysis identified additional KRAS G12/13 mutation positive patient in whom KRAS mutations were undetected by the tumor test suggesting tumor heterogeneity and/or inadequate biopsy. The dynamics of ctDNA KRAS mutational load in urine correlated with radiographic responses, especially in liver-dominant metastatic patients, suggesting that urinary ctDNA may be a valuable method for monitoring treatment responses in patients with mCRC. Citation Format: Afsaneh Barzi, Vlada Melnikova, Cecile Rose T. Vibat, Saege Hancock, Yan Ning, Dana Agafitei, Mark G. Erlander, Heinz-Josef Lenz. Use of urinary circulating tumor DNA (ctDNA) KRAS for monitoring treatment response in patients with metastatic colorectal cancer (mCRC). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B4.

Abstract 5240: Comparative levels of KRAS mutations circulating tumor DNA for association with overall survival in patients with non-resectable pancreatic cancer

Abstract Background: The median overall survival (OS) time of patients with non-resectable pancreatic cancer varies widely. Diagnostic tools are presently lacking to predict patient outcome at diagnosis. The vast majority of pancreatic tumors harbor KRAS mutations. In this study, we evaluated whether quantitative baseline and longitudinal monitoring of KRAS mutations in plasma circulating tumor DNA (ctDNA) may be used to stratify patients for predicting outcome. Methods: The Danish BIOPAC study prospectively collected plasma from patients with non-resectable pancreatic cancer undergoing treatment with gemcitabine or FOLFIRINOX. Archival (3-5 years) plasma specimens were collected from 113 patients pre-treatment (baseline),on chemotherapy, as well as at multiple additional time intervals for up to 977 days from baseline. Interim analysis of ctDNA KRAS was conducted (after 105 deaths). Levels of ctDNA KRAS mutations were assessed in 35 patients with long OS (median 473 days; range 360-1134), 33 patients with medium OS (median 227 days; range 155-349) and 37 patients with short OS (median 94 days; range 21-146). PCR enrichment of KRAS G12A/C/D/R/S/V, and G13D mutations was performed, followed by massively parallel deep sequencing and quantification with standardization of reporting number of copies detected per 105 genome equivalents (GE). Results: In a prospective-retrospective biomarker study of 113 patients, interim analysis of ctDNA KRAS was conducted (after 105 deaths). 92 of 105 patients had evaluable baseline plasma samples. Number of mutant KRAS copies was higher in patients with short OS (median 661; range 0-190,490 copies/105 GE) versus with median OS (median 103; range 0 to 275,918 copies/105 GE) versus with long OS (median, 15; range, 0-1,369 copies/105 GE). Longitudinally, KRAS mutation levels remained mostly low with long OS (last time point median 9; range 0-70,451 copies/105 GE) vs. medium OS (median 155; range 0-314,103 copies/105 GE) or short OS where levels increased or remained high (median 803; range 0-138,508 copies/105 GE). As this dramatic difference in systemic KRAS levels may reflect distinct tumor phenotypes, the underlying tumor biology was further investigated by interrogating additional cancer mutational hotspots (using massively parallel deep sequencing) in plasma ctDNA of patients stratified by systemic KRAS and the OS. Conclusion: Shorter OS in patients with non-resectable pancreatic cancer tended to associate with high levels of ctDNA KRAS mutations at diagnosis and with post-treatment elevation of KRAS mutations. ctDNA KRAS mutation levels in patients with non-resectable pancreatic cancer observed at diagnosis or on treatment may predict patient outcome and could reflect distinct underlying tumor biology. Citation Format: Julia S. Johansen, Cecile Rose T. Vibat, Saege Hancock, Latifa Hassaine, Errin Samuelsz, Inna Chen, Eric A. Collisson, Dan Calatayud, Benny V. Jensen, Jane Preuss Hasselby, Timothy T. Lu, Jason C. Poole, Vlada Melnikova, Mark G. Erlander. Comparative levels of KRAS mutations circulating tumor DNA for association with overall survival in patients with non-resectable pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5240. doi:10.1158/1538-7445.AM2015-5240

Detection of K‐ras gene mutation by liquid biopsy in patients with pancreatic cancer

Cell-free circulating tumor DNA (ctDNA) in serum has been considered to be a useful candidate for noninvasive cancer diagnosis. The current study was designed to estimate the clinical usefulness of genetic analysis for ctDNA by digital polymerase chain reaction in patients with pancreatic cancer. The authors compared K-ras mutations detected in endoscopic ultrasound-guided fine-needle aspiration biopsy tissue DNA and in ctDNA from 75 patients with pancreatic cancer. K-ras mutations in the serum of 66 independent, consecutive patients with pancreatic cancer were also analyzed and the authors compared the results with survival rates. The frequencies of the mutations in tissue samples at G12V, G12D, and G12R in codon 12 were 28 of 75 samples (37.3%), 22 of 75 samples (29.3%), and 6 of 75 samples (8.0%), respectively. Conversely, the rates of the mutations in ctDNA were 26 of 75 samples (34.6%), 29 of 75 samples (38.6%), and 4 of 75 samples (5.3%), respectively. Overall, the K-ras mutation rates in tissue and ctDNA were 74.7% and 62.6%, respectively, and the concordance rate between them was 58 of 75 samples (77.3%). Survival did not appear to differ by the presence of K-ras mutations in tissue DNA, but the survival of patients with K-ras mutations in ctDNA was significantly shorter than that of patients without mutations in both a development set (P = .006) and an independent validation set (P = .002). The difference was especially evident in cases with a G12V mutation. Analysis of ctDNA is a new useful procedure for detecting mutations in patients with pancreatic cancer. This noninvasive method may have great potential as a new strategy for the diagnosis of pancreatic cancer as well as for predicting survival.

Abstract 1736: Digital castPCR for direct detection of circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) in peripheral blood of lung cancer patients

Abstract [OBJECTIVES] Genetic alternations of EGFR and KRAS are frequently found in lung cancer cells. Accurate detection of these mutations from lung cancer peripheral blood may provide a noninvasive means for early cancer detection and disease monitoring. The goal of this study is to develop a quantitative analysis of EGFR and KRAS mutations both of ctDNA in plasma and of CTCs in blood cells from lung cancers by digital castPCR (competitive allele specific TaqMan-base PCR) technology. [METHODS] Molecular analysis and enumeration of CTCs in peripheral blood cells were analyzed by combining sample partition and digital castRT-qPCR assays without prior biophysical processing. The castPCR can detect rare copies of mutant alleles with a 6-log dynamic range and &amp;lt; 5-copy detection sensitivity for EGFR and KRAS mutations. Quantitative analysis of EGFR and KRAS mutations in ctDNA was done in OpenArray platform by digital castPCR. For CTC detection, whole blood samples with spiked-in known mutation and gen marker lung cancer cell lines were partitioned onto 96- or 384-well plate(s), such that each well contains either one cancer cell or none together with normal white blood cells and red blood cells. RNAs and/or DNAs were extracted by magnetic beads and were pre-amplified prior to molecular detection. Genetic mutations and cell type specific markers (CK19) for CTC identification and enumeration were determined by castRT-qPCR and TaqMan Gene expression (GEx) assays, respectively. Blood samples from lung cancer patients were first separated into blood cells and plasma portions before CTC detections. The sample plasma portions were used to detect EGFR and KRAS mutations of ctDNA by digital castPCR in OpenArray platform. [RESULTS] The sample partition process resulted in a relative CTC enrichment or digital enrichment of 20 - 400 folds (the relative ratio of CTC to normal cells) in a CTC-positive well. The RT-castPCR and TaqMan GEx assays accurately quantify the number of spiked-in tumor cells (10 - 60 cells/mL) in whole blood with known EGFR and/or KRAS mutations and CK19. For 10 blood samples from lung cancer patients, CTCs were detected from all patients even with stage I and II of 3 patients. In the plasma portions of the same samples, digital castPCR by OpenArray was able to detect EGFR mutations (p.L858R) and 19 mutations of EFGR exon 9. The results of EGFR mutations are consistent between intact CTCs and ctDNA. [CONCLUSION] Our preliminary data suggest that combination of digital sample enrichment and castRT-qPCR can be used to directly enumerate CTCs and detect cancer-related mutations in blood cells without prior biophysical sample enrichment. Digital castPCR by OpenArray can be used to assess circulating tumor DNA. Further test in larger clinical samples are warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1736. doi:1538-7445.AM2012-1736

Abstract LB-422: Quantitative detection of circulating tumor DNA by droplet-based digital PCR

Abstract Circulating tumor DNA (ctDNA) is present in plasma of individuals with advanced cancers. ctDNA is a prognostic marker for patients with colorectal cancer (CRC) and it might also be used for predicting the response to targeted therapy. For example, mutations in KRAS indicate which patients will fail to respond to specific therapies (cetuximab, panitunimab). Although ctDNA is characterized by the presence of a somatic mutation, direct quantitative detection through a simple workflow of such mutant DNA is not feasible by current technologies because the ratio of ctDNA to wild-type DNA can be as low as 1/10,000. This study describes the use of droplet-based digital PCR for detection and quantitation of one of the seven most frequent KRAS mutations in ctDNA from plasma of patients with advanced colorectal cancer. Furthermore, we demonstrate that multiplex digital PCR enables testing samples for different mutations simultaneously. By segregating individual target DNA molecules into millions of aqueous droplets acting as independent microreactors, our procedure allows for extremely precise, sensitive, and fast quantification of mutated genes. The sensitivity of the procedure was confirmed by measuring 1/200,000 dilution of KRAS-mutated cell-line DNA in a background of wild-type DNA. Furthermore, plasma of 14 patients with metastatic CRC were tested. Our procedure enabled detection of the target mutation and the wild-type DNA, and thus, measurement of the total amplifiable DNA. DNA concentration in the plasma samples varied by two orders of magnitude and was not correlated with the proportion of mutated DNA, which varied from 42% to 0.5%. By using a duplex analysis to detect either of the two most frequent mutations of KRAS (G12D and G13D) and the wild-type DNA, the expected mutation (known by primary tumor characterization) was detected in 12 out of the 14 samples. Two samples had a low amount of amplifiable DNA leading to an inconclusive result. Five samples, positive for the G13D mutation, were also tested for the G12D mutations and were negative. Moreover, we tested 4 samples in a multiplex format allowing the simultaneous analysis of wild-type, G12D, G13D, and G12R mutant KRAS. The multiplex testing results (i.e., the identity of the mutation and the mutant-to-wild-type ratio) were identical to those obtained in the duplex analysis. Our results demonstrate that our digital PCR method enables non-invasive detection of KRAS in plasma of patients with metastatic CRC with high sensitivity and high specificity. We anticipate that the method will be employed in multiple applications in the clinic, including diagnosis, cancer recurrence monitoring, and treatment management. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-422. doi:1538-7445.AM2012-LB-422