Detection Of RAS Mutations Research Articles

Early changes in circulating tumor DNA (ctDNA) predict treatment response in metastatic KRAS-mutated colorectal cancer (mCRC) patients

The detection of RAS mutations and co-mutations in liquid biopsy offers a novel paradigm for the dynamic management of metastatic colorectal cancer (mCRC) patients.Expanding the results of the prospective OMITERC (OMIcs application from solid to liquid biopsy for a personalized ThERapy of Cancer) project, we collected blood samples at specific time points from patients who received a first-line chemotherapy (CT) for KRAS-mutated mCRC. CTC quantification was performed by CellSearch® system. Libraries from cfDNA were prepared using the Oncomine™ Colon cfDNA Assay to detect tumour-derived DNA in cfDNA. The analysis involved >240 hotspots in 14 genes.Twenty patients with KRAS-mutated mCRC treated at the Medical Oncology Unit of Careggi University Hospital were prospectively enrolled. Nine patients had available data for longitudinal monitoring of cfDNA. After 6 weeks of first-line CT an increase of KRAS-mutated clone was reported in the only patient who did not obtain disease control, while all patients with decrease of KRAS clones obtained disease control. Overall, in patients with a short (<9 months) progression-free survival (PFS) we registered, at 6 weeks, an increase in cfDNA levels and in KRAS mutations or other co-mutations, i.e. PIK3CA, FBXW7, GNAS, and TP53. In selected cases, co-mutations were able to better anticipate radiological progressive disease (PD) than the increase of KRAS-mutated clones.In conclusion, our study confirms plasma ctDNA as a crucial tool for anticipating PD at an early time point and highlights the value of a comprehensive assessment of clonal dynamics to improve the management of patients with mCRC.

Novel Clinical Tool to Estimate Risk of False-Negative KRAS Mutations in Circulating Tumor DNA Testing.

In metastatic colorectal cancer, the detection of RAS mutations by circulating tumor DNA (ctDNA) has emerged as a valid and noninvasive alternative approach to determining RAS status. However, some RAS mutations may be missed, that is, false negatives can occur, possibly compromising important treatment decisions. We propose a statistical model to assess the probability of false negatives when performing ctDNA testing for RAS. Cohorts of 172 subjects with tissue and multipanel ctDNA testing from MD Anderson Cancer Center and 146 subjects from Massachusetts General Hospital were collected. We developed a Bayesian model that uses observed frequencies of reference mutations (the maximum of APC and TP53) to provide information about the probability of KRAS false negatives. The model was alternatively trained on one cohort and tested on the other. All data were collected on Guardant assays. The model suggests that negative KRAS findings are believable when the maximum of APC and TP53 frequencies is at least 8% (corresponding posterior probability of false negative <5%). Validation studies demonstrated the ability of our tool to discriminate between false-negative and true-negative subjects. Simulations further confirmed the utility of the proposed approach. We suggest clinicians use the tool to more precisely quantify KRAS false-negative ctDNA results when at least one of the reference mutations (APC, TP53) is observed; usage may be especially important for subjects with a maximum reference frequency of <8%. Extension of the methodology to predict false negatives of other genes is possible. Additional reference genes can also be considered. Use of personal training data sets is supported. An open-source R Shiny application is available for public use.

Detection of ras mutations leads to non-aggressive treatment of thyroid tumors

Detection of ras mutations leads to non-aggressive treatment of thyroid tumors

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.

MicroRNA expression profiling of RAS-mutant thyroid tumors with follicular architecture: microRNA signatures to discriminate benign from malignant lesions

PurposeRAS mutations represent common driver alterations in thyroid cancer. They can be found in benign, low-risk and malignant thyroid tumors with follicular architecture, which are often diagnosed as indeterminate nodules on preoperative cytology. Therefore, the detection of RAS mutations in preoperative setting has a suboptimal predictive value for malignancy. In this study, we investigated differentially expressed microRNA (miRNA) in benign and malignant thyroid tumors with follicular architecture carrying mutations in RAS genes.MethodsTotal RNA was purified from 60 RAS-mutant follicular-patterned thyroid tumors, including follicular adenoma (FA), noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP), papillary and follicular thyroid carcinoma cases (PTC, FTC); 22 RAS-negative FAs were used as controls. The expression analysis of 798 miRNAs was performed by digital counting (nCounter nanoString platform).ResultsComparing RAS-mutant and RAS-negative FAs, 12 miRNAs showed significant deregulation, which was likely related to the oncogenic effects of RAS mutations. Twenty-two miRNAs were differentially expressed in RAS-mutant benign versus malignant tumors. Considering the tumor type, 24 miRNAs were deregulated in PTC, 19 in NIFTP, and seven in FTC and compared to FA group; among these, miR-146b-5p, miR-144-3p, and miR-451a showed consistent deregulation in all the comparisons with the highest fold change.ConclusionsThe miRNA expression analysis of follicular-patterned thyroid tumors demonstrated that RAS mutations influences miRNA profile in benign tumors. In addition, several miRNAs showed a histotype-specific deregulation and could discriminate between RAS-mutant benign and RAS-mutant malignant thyroid lesions, thus deserving further investigation as potential diagnostic markers.

KRAS and NRAS mutational analysis in plasma ctDNA from patients with metastatic colorectal cancer by real-time PCR and digital PCR.

Mutations in the KRAS and NRAS (RAS) genes are negative predictors of response to anti-EGFR therapy in metastatic colorectal cancer (mCRC). The detection of mutations in circulating tumor DNA (ctDNA) has emerged as a less invasive strategy to assess the molecular profile of mCRC patients. We aimed to perform RAS mutational analysis in ctDNA from mCRC patients using BEAMing Digital PCR (OncoBEAM) and Idylla ctDNA qPCR and evaluate the concordance rate with RAS mutational status in tumor tissue and between these two methodologies with different limits of detection. Blood samples were collected from 47 mCRC patients previously tested for RAS mutations in tumor tissue. DNA was extracted from plasma using the QIAamp Circulating Nucleic Acid Kit, and RAS mutation analysis was conducted using OncoBEAM RAS CRC and Idylla ctRAS assays. The overall agreement between tumor tissue and ctDNA analyses was 83% and 78.7% using the OncoBEAM and Idylla assays, respectively, with the concordance being 96.2% and 88.5% in naive treatment patients. The overall agreement between OncoBEAM and Idylla ctDNA analyses was 91.7%. Analysis of ctDNA is a viable strategy for clinical management of mCRC patients. Although the OncoBEAM assay sensitivity is somewhat higher, the fully automated Idylla platform also has good performance, while being cheaper and much less labor-intensive, for the detection of RAS mutations in plasma, either at diagnosis or after progression when considering anti-EGFR treatment rechallenge.

Rationale and Study Design of the PARERE Trial: Randomized phase II Study of Panitumumab Re-Treatment Followed by Regorafenib Versus the Reverse Sequence in RAS and BRAF Wild-Type Chemo-Refractory Metastatic Colorectal Cancer Patients

BackgroundA recent phase II randomized Japanese study reported better survival with regorafenib followed at progression by cetuximab ± irinotecan compared with the reverse standard sequence in chemo-refractory and anti-EGFR-naïve, RAS wild-type (wt) mCRC patients. Nowadays the use of anti-EGFR antibodies is more frequently anticipated to the first-line of therapy especially in patients with left-sided RAS/BRAF wt tumours. However, retrospective analyses and phase II single-arm trials showed promising activity of re-using anti-EGFRs in metastatic colorectal cancer (mCRC) patients who previously achieved benefit from a first-line anti-EGFR-based treatment. Post-hoc analyses of these trials revealed that the detection of RAS mutations in circulating tumour DNA (ct-DNA) at the time of re-treatment may be useful to identify resistant patients. Patients and MethodsPARERE (NCT04787341) is a prospective, open label, multicentre phase II study in which 214 RAS/BRAF wt chemo-refractory mCRC patients with previous benefit from first-line anti-EGFR-based treatment and RAS/BRAF wt ct-DNA in the liquid biopsy collected at the time of inclusion will be randomized in a 1:1 ratio to receive panitumumab followed after progression by regorafenib versus the reverse sequence. Primary endpoint is overall survival. Secondary endpoints are 1st-progression free-survival (PFS), 2nd-PFS, time to failure strategy, objective response rate, and safety. Aim of the studyThe aim of this study is to validate the role of anti-EGFR retreatment and its proper placement in the therapeutic route of mCRC patients selected according to the analysis of ct-DNA in liquid biopsy. Results are expected at the end of 2023.

A novel clinical tool to estimate risk of false negative KRAS mutation in circulating tumor DNA testing.

3594 Background: Recently, in metastatic colorectal cancer (mCRC), the detection of RAS mutations by circulating tumor (ct) DNA has recently emerged as a valid and non-invasive alternative approach, overall showing a high concordance with the standard tissue genotyping, giving information on response to EGFRi treatment and resistant mechanisms. However, RAS mutations may be missed due to low levels of any ctDNA in the blood (false-negative), and it has been difficult to distinguish this from patients without a RAS mutation in the tumor (true-negative). We propose a methodology that can be applied to multi-gene ctDNA testing panels to accurately distinguish true- and false-negative tests. Methods: 357 subjects with tissue and multi-panel ctDNA testing from MD Anderson (MDACC) were used as a training dataset and 295 subjects from Massachusetts General Hospital (MGH) dataset as the testing dataset. CtDNA panels contained between 65 and 70 genes, allowing evaluation of tumor ctDNA shedding from variant allele fraction (VAF) levels in the plasma from other genes (such as APC and TP53). Based on the relationship between KRAS and the VAFs of other gene, we established a Bayesian model providing a posterior probability of false negative in the ctDNA test, using thresholds of &lt; 5% (low), 5-15% (medium), and &gt; 15% (high). This model was validated on the MGH database. Results: Across both cohorts, 431 patients were ctDNA wild type for KRAS. Of those, 29 had tissue documenting a KRAS mutation for a false negative rate of 8%. The model provides the posterior probability that a KRAS mutation is indeed present in the tissue given the observed values of allele frequencies for other mutated genes in the plasma. In the validation cohort, a predicted low false negative had no false negatives (0/62, 95% CI 0%-5.8%), while a predicted medium false negative rate was associated with 3% false negative (1/32, 95% CI 0%-16%). In contrast, a high predicted false negative rate was associated with 5% false negative (5/100, 95% CI 1.6%-11%). The results demonstrate the ability of our tool to discriminate between subjects with true negative and false negatives, as a higher proportion of false negatives are observed at higher posterior probabilities. Conclusions: In conclusion, our approach provides increased confidence in KRAS ctDNA mutation testing in clinical practice, thereby facilitating the identification patients who will benefit from EGFR inhibition while reducing the risk of false negative tests. Extension of this methodology to NRAS and BRAF is possible, with clinical application enabled by a freely available online tool.

True conversions from RAS mutant to RAS wild-type in circulating tumor DNA from metastatic colorectal cancer patients as assessed by methylation and mutational signature.

The paucity of targeted treatments available in patients with RAS mutant colorectal cancers contributes to the poor prognosis of this patient group compared to those with RAS wild-type disease. Recent liquid biopsy-driven studies have demonstrated that RAS mutant clones might disappear in plasma during the clonal evolution of the disease, opening new unforeseen perspectives for EGFR blockade in these patients. Nevertheless, the lack of detection of RAS mutations in plasma might depend on the low amount of released circulating tumor DNA (ctDNA), making it necessary a more accurate selection of patients with true RAS mutation conversions. In this liquid biopsy-based study, we assessed RAS mutational status in initially RAS-mutant patients at the time of progressive disease from any line of therapy and investigated the incidence of true conversions to plasma RAS wild-type, comparing a colon cancer specific methylation profile with a mutational signature of ctDNA. Globally, considering either mutational panel or methylation profile as reliable tests to confirm or exclude the presence of ctDNA, the percentage of "true RAS converters" was 37.5%. In our series we observed a trend toward a better PFS in patients who received anti-EGFR as second or subsequent treatment lines compared to those who did not.

KRAS and NRAS mutation detection in circulating DNA from patients with metastatic colorectal cancer using BEAMing assay: Concordance with standard biopsy and clinical evaluation.

Patients with metastatic colorectal cancer (mCRC) are routinely screened for either K- and N-RAS to select the appropriate treatment. The present study aimed to evaluate the concordance between K- and NRAS status in the tissue (either primary tumor or metastasis) and the plasma of patients with mCRC and to identify the associations between K- and NRAS mutations in ctDNA and the clinicopathological parameters. Samples from a total of 31 patients with mCRC with measurable disease according to the Response Evaluation Criteria in Solid Tumors were analyzed. For all patients, K- and NRAS status was determined in the tissue by matrix-assisted laser desorption/ionization time of flight mass spectrometry. For the detection of RAS mutations in cell-free tumor DNA also defined as circulating tumor DNA (ctDNA), the OncoBEAM® RAS CRC kit (Sysmex Inostics) was used. A total of 6/31 tissue samples expressed wild-type KRAS, whereas 25/31 presented mutations. In addition, 7/31 plasma samples expressed wild-type KRAS, mutations were detected in 22/31 patients, and for 2/31 patients, the test did not provide a conclusive result. A total of 24/31 patients expressed wild-type NRAS, 6/31 had mutations and 1/21 was not informative. For the KRAS mutational status, a moderate concordance (agreement, 85.18%; Cohen's k, 0.513) between the tissue and plasma analysis was observed; for NRAS, a fair agreement (agreement, 83.33%; Cohen's k, 0.242) was obtained. In conclusion, both tissue and plasma analyses should be performed for the management of patients with mCRC. To better exploit the beads, emulsions, amplification, magnetics (BEAMing) technique in the clinical setting, studies aimed at determining the RAS status to monitor therapy and during follow-up are warranted.

Diffusion-Weighted MRI and Diffusion Kurtosis Imaging to Detect RAS Mutation in Colorectal Liver Metastasis.

To detect RAS mutation in colorectal liver metastasis by Diffusion-Weighted Magnetic Resonance Imaging (DWI-MRI) - and Diffusion Kurtosis imaging (DKI)-derived parameters. In total, 106 liver metastasis (60 metastases with RAS mutation) in 52 patients were included in this retrospective study. Diffusion and perfusion parameters were derived by DWI (apparent diffusion coefficient (ADC), basal signal (S0), pseudo-diffusion coefficient (DP), perfusion fraction (FP) and tissue diffusivity (DT)) and DKI data (mean of diffusion coefficient (MD) and mean of diffusional Kurtosis (MK)). Wilcoxon-Mann-Whitney U tests for non-parametric variables and receiver operating characteristic (ROC) analyses were calculated with area under ROC curve (AUC). Moreover, pattern recognition approaches (linear classifier, support vector machine, k-nearest neighbours, decision tree), with features selection methods and a leave-one-out cross validation approach, were considered. A significant discrimination between the group with RAS mutation and the group without RAS mutation was obtained by the standard deviation value of MK (MK STD), by the mean value of MD, and by that of FP. The best results were reached by MK STD with an AUC of 0.80 (sensitivity of 72%, specificity of 85%, accuracy of 79%) using a cut-off of 203.90 × 10-3, and by the mean value of MD with AUC of 0.80 (sensitivity of 84%, specificity of 73%, accuracy of 77%) using a cut-off of 1694.30 mm2/s × 10-6. Considering all extracted features or the predictors obtained by the features selection method (the mean value of S0, the standard deviation value of MK, FP and of DT), the tested pattern recognition approaches did not determine an increase in diagnostic accuracy to detect RAS mutation (AUC of 0.73 and 0.69, respectively). Diffusion-Weighted imaging and Diffusion Kurtosis imaging could be used to detect the RAS mutation in liver metastasis. The standard deviation value of MK and the mean value of MD were the more accurate parameters in the RAS mutation detection, with an AUC of 0.80.

Circulating Cell-Free DNA-Based Liquid Biopsy Markers for the Non-Invasive Prognosis and Monitoring of Metastatic Pancreatic Cancer.

Liquid biopsy may assist in the management of cancer patients, which can be particularly applicable in pancreatic ductal adenocarcinoma (PDAC). In this study, we investigated the utility of circulating cell-free DNA (cfDNA)-based markers as prognostic tools in metastatic PDAC. Plasma was obtained from 61 metastatic PDAC patients, and cfDNA levels and fragmentation were determined. BEAMing technique was used for quantitative determination of RAS mutation allele fraction (MAF) in cfDNA. We found that the prognosis was more accurately predicted by RAS mutation detection in plasma than in tissue. RAS mutation status in plasma was a strong independent prognostic factor for both overall survival (OS) and progression-free survival (PFS). Moreover, RAS MAF in cfDNA was also an independent risk factor for poor OS, and was strongly associated with primary tumours in the body/tail of the pancreas and liver metastases. Higher cfDNA levels and fragmentation were also associated with poorer OS and shorter PFS, body/tail tumors, and hepatic metastases, whereas cfDNA fragmentation positively correlated with RAS MAF. Remarkably, the combination of CA19-9 with MAF, cfDNA levels and fragmentation improved the prognostic stratification of patients. Furthermore, dynamics of RAS MAF better correlated with patients’ outcome than standard CA19-9 marker. In conclusion, our study supports the use of cfDNA-based liquid biopsy markers as clinical tools for the non-invasive prognosis and monitoring of metastatic PDAC patients.

MON-514 Detection of RAS Mutations and RET/PTC Fusions in Thyroid Cancer Using Microfluidic Digital PCR

Background:The identification of somatic mutations and gene fusions is crucial for guiding therapeutic decisions in patients with thyroid cancer. Microfluidic digital PCR is currently considered as a technique of choice for highly sensitive detection of gene mutations/fusion. We recently demonstrated that dPCR is a useful tool for detection of BRAFV600E and TERT promoter mutations in thyroid tumors.Objectives:This study aimed to determine the analytic and clinical validity of dPCR for detection of RAS mutations and RET/PTC fusions in thyroid cancer tissue.Material and Methods:Thyroid tissues from 75 patients with PTCs (58 classical PTC (CPTC) and 17 follicular variant (FVPTC) were used for DNA and RNA extraction. The rare mutation SNP genotyping assays which were multiplexed for detection of mutant and wild type NRASQ61; as well as RET/PTC1 and RET/PTC3 were synthesized by Thermo Fisher Scientific. Digital PCR was performed using a QuantStudio 3D Digital PCR platform. QuantStudio Software was used for relative and quantitative data analysis.Results:NRASQ61 was detected in 0/58 CPTC and in 6/17 (35%) FVPTC. The ratios of mutant/total varying from 11.7% to 61.5%. Among patients with FVPTC there were no significant associations between the presence of NRASQ61 and patient’s age, sex, multifocal growth, extra-thyroidal invasion and lymph node metastases. The ratios mutant/total correlated with tumor size in patients harboring NRASQ61. In 23 cases, RET/PTC1 and RET/PTC3 transcripts were examined. RET/PTC1 and RET/PTC3 transcripts were detected in 3 and 1 case, respectively. RET/PTCs were detected in CPTC, but not in FVPTC. RET/PTC positive tumors were characterized by multi-focal patterns of growth, presence of extra-thyroidal invasion, and presence of lymph node metastases (4 of 4 cases with RET/PTC). There were not RET/PTCs positive tumors harboring simultaneously anomalies in RAS oncogene.Conclusions:Microfluidic digital PCR allows specific, sensitive and rapid detection of RAS mutations and RET/PTC fusions in thyroid tissue samples. Implementation of dPCR-based assays may facilitate analysis of thyroid tumors and support research in patients with thyroid cancer.

Concordance between blood- and tumor-based detection of RAS mutations to guide therapy in metastatic colorectal cancer.

217 Background: The study of RAS using circulating tumor DNA (ctDNA) provides a good alternative for the detection and monitoring of RAS mutations during therapy course. Liquid biopsy has the advantage of being less invasive than conventional tissue biopsy. Therefore, it is necessary to determine the degree of concordance of RAS status between plasma and tissue samples from mCRC patients. Methods: We conducted a study to evaluate RAS concordance in several hospitals in Galicia, Northwest of Spain. We analyzed samples from 380 patients with mCRC in Galicia. RAS genotyping in plasma was performed using OncoBEAM RAS CRC kit, Sysmex and compared with the PyroMark Q24 System, Qiagen for FFPE-tissue analysis. Clinical data were collected from electronical reports from each patient. We analyzed the clinical features that can be related with discordant cases. Results: The overall percent of agreement was 83%, with a positive concordance of 79% (143/180 patients) and a negative concordance of 87% (116/132 patients). Taking into account the clinical features of the patients, those with only liver metastasis showed a higher level of concordance (88,8%) and the highest level was in the group of patients that presented liver metastasis and another location (91,3%). On the other hand, patients with only peritoneal disease showed the lowest level of agreement (68,2%), and the absence of liver metastasis was a significant factor of discordance of RAS (71% vs 28%, p&lt;0.0001). Conclusions: The high overall concordance between plasma and tissue RAS mutation support the liquid biopsy technology as an alternative to tissue testing for RAS characterization in mCRC patients, especially in patients with liver metastasis and less in those with peritoneal disease. These results reinforce the use of liquid biopsy as a non-invasive tool for guiding targeted therapy in our patients. [Table: see text]

Plasma ctDNA RAS mutation analysis by digital polymerase chain reaction in patients with inoperable pancreatic cancer

Abstract Background Pancreatic ductal adenocarcinoma (PDAC) is associated with poor prognosis and high mortality worldwide. Despite the emergence of new therapeutic approaches for the treatment of inoperable PDAC, reliable prognostic/predictive biomarkers are lacking. PDAC harbours mutations in the KRAS gene in 80-90% of patients. The aim of the present study is the detection and quantification of RAS mutations in the ctDNA of patients with inoperable PDAC by BEAMing digital polymerase chain reaction (PCR), in order to (a) evaluate the correlation with tissue RAS mutation status in the tumour, (b) study the baseline prognostic significance and (c) study the predictive significance of ctDNA RAS mutation kinetics for the prediction of response to systemic/neoadjuvant therapy. Methods Plasma samples of patients with locally advanced or metastatic inoperable PDAC were collected at diagnosis and at 4-6 weeks after treatment initiation and were tested for 34 mutations in the KRAS and NRAS genes by BEAMing digital polymerase chain reaction (PCR). This method has a particularly high sensitivity and can accurately quantify mutated DNA as the Mutant Allele Fraction (MAF). To test the efficacy and validity of ctDNA for the detection of RAS mutations, RAS mutations will also be tested for on paired tumour tissue samples of the patients under study by PCR. Results So far, 38 plasma samples from 20 patients were available for analysis. At baseline, mutant ctDNA pertaining to exon 2 codon 12 of the KRAS gene was detected in all patients’ samples and, additionally, to exon 3 codon 61 of the NRAS gene in one of them. Comparison of plasma and tissue results revealed a high agreement of the RAS mutational profile in plasma ctDNA and tissue tumour DNA. Kinetics of the RAS mutational load during therapy and its prognostic or predictive value will be analysed with further cohort expansion. Conclusions This work highlights the potential role of liquid biopsies in the management of PDAC and, more specifically, will yield preliminary data on the role of RAS ctDNA profiling as a predictive and prognostic marker. Legal entity responsible for the study Department of Medical Oncology, University General Hospital of Ioannina. Funding Hellenic Society of Medical Oncology (HeSMO) and Society for Study of Clonal Heterogeneity of Neoplasia (EMEKEN). Disclosure G. Pentheroudakis: Research grant / Funding (institution), Support material was provided for this project: Sysmex Inostics. All other authors have declared no conflicts of interest.

Customisation of therapeutic strategy in metastatic colorectal cancer by use of liquid biopsies: Updated results of an observational study

Abstract Background Metastatic colorectal cancer (mCRC) affects 20% of patients diagnosed with colorectal malignancy and is a major cause of cancer-related death worldwide. Epidermal growth factor receptor (EGFR) blockade with monoclonal antibodies, in combination with chemotherapy, has been shown to benefit patients with RAS wild type tumours. Our project explores real-time assessment of mCRC mutational profile, by use of two methods of circulating tumour DNA (ctDNA) analysis, and its potential to better guide therapeutic decisions. Methods Baseline plasma samples were prospectively collected from 64 patients at diagnosis of mCRC and at disease progression. Analysis of KRAS and NRAS exons 2, 3 and 4 was performed with the highly sensitive BEAMing digital polymerase chain reaction (PCR) and next-generation sequencing (NGS). In a subgroup of patients, with baseline RAS mutations, mid-treatment liquid biopsies were performed, to assess the change in ctDNA RAS mutational load as response biomarker. Mutational status of matched tumour tissue, generated with a conventional PCR-based method or NGS, was prospectively recorded for all patients. Results Preliminary results of our study, presented at the EACR-ESMO Joint Conference on Liquid Biopsies, have shown detection of RAS mutations in the ctDNA from over 50% of patients, with a satisfactory plasma-tissue concordance. An update on these data, as well as on the predictive value of ctDNA RAS mutational load dynamics, will be presented. Disease progression data, in light of baseline RAS status, will also be revisited, as almost 20% of patients have progressed on first-line treatment. In addition, the concordance of BEAMing and NGS as liquid biopsy methods is to be studied in a subgroup of patients with available plasma samples. Conclusions The larger sample size and the maturity of this updated analysis further add on the value of our work, the results of which remain in line with the literature. Our study serves as a real-life example of the potential of liquid biopsies to detect RAS mutation status changes, allowing for precision-oncology therapeutic approaches in the continuum of care of metastatic colorectal cancer patients. Legal entity responsible for the study Department of Medical Oncology, University General Hospital of Ioannina. Funding Amgen, Hellenic Society of Medical Oncology (HeSMO) and Society for Study of Clonal Heterogeneity of Neoplasia (EMEKEN). Disclosure G. Pentheroudakis: Research grant / Funding (institution): Amgen. All other authors have declared no conflicts of interest.

RAS genes in colorectal carcinoma: pathogenesis, testing guidelines and treatment implications

The RAS family is among the most commonly mutated genes in all human malignancies including colon cancer. In normal cells, RAS proteins act as a link in the intracellular signal...

Detection of RAS mutations in circulating tumor DNA: a new weapon in an old war against colorectal cancer. A systematic review of literature and meta-analysis.

Background:Tissue evaluation for RAS (KRAS or NRAS) gene status in metastatic colorectal cancer (mCRC) patients represent the standard of care to establish the optimal therapeutic strategy. Unfortunately, tissue biopsy is hampered by several critical limitations due to its invasiveness, difficulty to access to disease site, patient’s compliance and, more recently, neoplastic tissue spatial and temporal heterogeneity.Methods:The authors performed a systematic literature review to identify available trials with paired matched tissue and ctDNA RAS gene status evaluation. The authors searched EMBASE, MEDLINE, Cochrane, www.ClinicalTrials.gov, and abstracts from international meetings. In total, 19 trials comparing standard tissue RAS mutational status matched paired ctDNA evaluated through polymerase chain reaction (PCR), next generation sequencing (NGS) or beads, emulsions, amplification and magnetics (BEAMing) were identified.Results:The pooled sensitivity and specificity of ctDNA were 0.83 (95% CI: 0.80–0.85) and 0.91 (95% CI: 0.89–0.93) respectively. The pooled positive predictive value (PPV) and negative predictive value (NPV) of the ctDNA were 0.87 (95% CI: 0.81–0.92) and 0.87 (95% CI: 0.82–0.92), respectively. Positive likelihood ratio (PLR) was 8.20 (95% CI: 5.16–13.02) and the negative likelihood ratio (NLR) was 0.22 (95% CI: 0.16–0.30). The pooled diagnostic odds ratio (DOR) was 50.86 (95% CI: 26.15–98.76), and the area under the curve (AUC) of the summary receiver operational characteristics (sROC) curve was 0.94.Conclusion:The authors’ meta-analysis produced a complete and updated overview of ctDNA diagnostic accuracy to test RAS mutation in mCRC. Results provide a strong rationale to include the RAS ctDNA test into randomized clinical trials to validate it prospectively.

550P - Evaluation of the sensitivity of RAS mutation detection of the Idylla platform in comparison to the OncoBEAM RAS CRC assay

550P - Evaluation of the sensitivity of RAS mutation detection of the Idylla platform in comparison to the OncoBEAM RAS CRC assay

Targeted data-independent acquisition for mass spectrometric detection of RAS mutations in formalin-fixed, paraffin-embedded tumor biopsies

Genomic testing for KRAS and NRAS mutations in clinical biopsies of various cancers is routinely performed to predict futility of anti-epidermal growth factor receptor (anti-EGFR) therapies. We hypothesized that RAS mutations could be detected and quantified at the protein level for diagnostic purposes using data-independent acquisition (DIA)-based mass spectrometry in formalin-fixed, paraffin-embedded (FFPE) tumor samples. We developed a targeted DIA assay that surveys the specific mass range of all possible peptides harboring activating mutations in KRAS exon 2. When the assay was applied to tumor samples with known KRAS or NRAS mutations (G12A, G12D, G12V, and G13D), RAS-mutant and wild-type peptides were successfully detected in 11 of 13 biopsy samples. Mutation statuses obtained by DIA were concordant with those obtained by DNA sequencing, and yields of mutant peptide (mutant peptide/[mutant + wild-type peptides]) exhibited linear correlation with yields of RAS-mutant mRNA. When applied to biopsy samples with failed DNA testing results, the DIA assay identified an additional RAS-mutated sample. SignificanceProteomic detection of RAS mutations by DIA in tumor biopsies can provide solid evidence of mutant RAS protein regardless of the mutation types and sites in exon 2. This robust method could rescue samples that fail genomic testing due to insufficient tumor tissue or lack of sequenceable DNA. It can be used to explore the relationship between protein expression level of mutant RAS and therapeutic outcome.