KRAS Mutations In Tissue Research Articles

The Prognostic Utility of KRAS Mutations in Tissue and Circulating Tumour DNA in Colorectal Cancer Patients

This study aims to investigate the long-term prognostic utility of circulating tumour DNA (ctDNA) KRAS mutations in colorectal cancer (CRC) patients and compare this with KRAS mutations in matched tissue samples. Tumour tissue (n = 107) and ctDNA (n = 80) were obtained from patients undergoing CRC resection and were analysed for KRAS mutations. The associations between KRAS mutation and overall survival (OS), cancer-specific survival (CSS), and recurrence-free survival (RFS) were analysed. All outcomes were measured in years (y). A total of 28.8% of patients had KRAS mutations in ctDNA and 72.9% in tumour tissue DNA. The high frequency of KRAS mutations in tissue samples was due to 51.4% of these being a detectable low mutation allele frequency (<10% MAF). Comparing KRAS mutant (KRASmut) to KRAS wild-type (KRASwt) in ctDNA, there was no association found with OS (mean 4.67 y vs. 4.34 y, p = 0.832), CSS (mean 4.72 y vs. 4.49 y, p = 0.747), or RFS (mean 3.89 y vs. 4.26 y, p = 0.616). Similarly, comparing KRASmut to KRASwt in tissue DNA there was no association found with OS (mean 4.23 y vs. 4.61 y, p = 0.193), CSS (mean 4.41 y vs. 4.71 y, p = 0.312), or RFS (mean 4.16 y vs. 4.41 y, p = 0.443). There was no significant association found between KRAS mutations in either tissue or ctDNA and OS, CSS, or RFS.

Analysis of Circulating DNA to Assess Prognoses for Metastatic Colorectal Cancer Patients Treated with Regorafenib Dose-Escalation Therapy: A Retrospective, Exploratory Analysis of the RECC Trial

Introduction: Regorafenib is a multi-kinase inhibitor approved for patients with metastatic colorectal cancer (mCRC) who were previously treated with standard therapies. A few reports showed the impact of KRAS mutation on therapeutic efficacy of regorafenib. Only one study reported poor prognoses for patients treated with regorafenib who had large amounts of circulating cell-free DNA (cfDNA). In the present study, we evaluated the impact of KRAS mutations in tissue or plasma and amounts of cfDNA on prognoses of mCRC patients treated with regorafenib. Method: This is a biomarker investigation of the RECC study, which evaluated efficacy of regorafenib dose-escalation therapy. Plasma samples were obtained just before initiation of treatment with regorafenib. KRAS mutations were evaluated using tissue and plasma samples. cfDNA was extracted from plasma samples and quantified. Results: Forty-five patients were enrolled in this biomarker study. Median progression-free survival (PFS) and overall survival (OS) of patients without KRAS mutations in tissues were 1.9 months (95% confidence interval [CI] 1.7–2.0) and 8.9 months (95% CI: 6.5–11.2), and those of patients with KRAS mutations were 1.4 months (95% CI: 1.3–1.5) and 6.8 months (95% CI: 5.0–8.5). Median PFS and OS of patients with plasma KRAS mutations were 1.9 months (95% CI: 1.8–1.9) and 7.0 months (95% CI: 5.3–8.7), respectively. Median PFS and OS of patients without plasma KRAS mutations were 1.7 months (95% CI: 1.1–2.3) and 8.9 months (95% CI: 6.7–11.2), respectively. Prior to administration of regorafenib, KRAS mutations were detected in 6 of 16 (37.5%) patients who had no tissue KRAS mutations. Median OS of patients with high cfDNA concentration (>median) was significantly poorer than that of patients with low cfDNA. Conclusion: KRAS mutations in the tissue or plasma have no impact on efficacy of regorafenib. KRAS emerging mutations were observed in quite a few patients. Large amounts of cfDNA may indicate poorer prognoses for patients receiving late-line regorafenib chemotherapy.

Diagnostic accuracy of circulating free DNA testing for the detection of KRAS mutations in non-small cell lung cancer: A systematic review and meta-analysis.

Kirsten rat sarcoma viral oncogene homolog (KRAS) gene encodes a GTPase that acts as a molecular switch for intracellular signal transduction, promoting cell growth and proliferation. Mutations in the KRAS gene represent important biomarkers for NSCLC targeted therapy. However, detection of KRAS mutations in tissues has shown some limitations. During the last years, analyses of circulating free DNA (cfDNA) has emerged as an alternative and minimally invasive, approach to investigate tumor molecular changes. Here, we assessed the diagnostic performance of cfDNA analysis, compared to tissues through a meta-analysis and systematic review of existing literature. From 561 candidate papers, we finally identified 40 studies, including 2,805 NSCLC patients. We extracted values relating to the number of true-positive, false-positive, false-negative, and true-negative. Pooled sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, and diagnostic odds ratio, each with 95% CI, were calculated. A summary receiver operating characteristic curve and the area under curve (AUC) were used to evaluate the overall diagnostic performance. The pooled sensitivity was 0.71 (95% CI 0.68-0.74) and the specificity was 0.93 (95% CI 0.92-0.94). The diagnostic odds ratio was 35.24 (95% CI 24.88-49.91) and the area under the curve was 0.92 (SE = 0.094). These results provide evidence that detection of KRAS mutation using cfDNA testing is of adequate diagnostic accuracy thus offering to the clinicians a new promising screening test for NSCLC patients.

Abstract 519: Clinical impact ofKRASG12, G13, Q61, K117 and A146 mutations in patients with colorectal liver metastases

Abstract Introduction: The distribution of KRAS mutation variants across tumor types is not uniform. The KRAS A146 mutation is predominantly seen in colorectal cancer (CRC) patients. Here, we evaluated how clinical features like tumor load and overall survival differ between metastatic CRC (mCRC) patients carrying distinct somatic KRAS G12, G13, Q61, K117 or A146 mutations. Methods: 419 CRC patients with initially unresectable liver-limited metastases, who participated in the multicenter CAIRO5 prospective clinical trial, were evaluated for tumor tissue KRAS mutation status. For the subgroup of patients who carried a KRAS mutation and were treated with bevacizumab and doublet or triplet chemotherapy (N=156), clinical outcome was evaluated and pretreatment tumor burden was quantified as liquid biopsy circulating tumor DNA (ctDNA) mutant allele fraction (MAF) and as total tumor volume (TTV) on CT imaging. The MAF, TTV and overall survival were compared between patients harboring different KRAS mutation variants. Results: Of the 156 patients with a KRAS mutated tumor, most carried a KRAS G12 mutation (N=112, 71.8%), followed by mutations in G13 (N=15, 9.6%), A146 (N=12, 7.7%), Q61 (N=9, 5.8%) and K117 (N=5, 3.2%). High plasma ctDNA levels were observed for patients carrying a KRAS A146 mutation versus those with a KRAS G12 mutation, with a median MAF of 48% versus 19%, respectively. Radiological TTV revealed this difference to be associated with a higher tumor load in patients harboring a KRAS A146 mutation (median TTV 672 cm3 (A146) versus 74 cm3 (G12), p=0.036). Moreover, KRAS A146 mutation carriers showed inferior overall survival compared to patients with mutations in KRAS G12 (median 10.7 versus 26.4 months; HR=2.5; p=0.003), and the multivariable Cox regression analysis showed that the KRAS alteration was the only independent prognostic factor for overall survival. Conclusion and Relevance: This study revealed that within mCRC patients A146 is the third most common KRAS mutation variant, and mCRC patients carrying a KRAS A146 mutation represent a distinct molecular subtype of patients with high tumor burden and poor clinical outcome. This highlights the importance of testing CRC for all KRAS mutations in routine clinical care and shows the opportunity for personalized treatment beyond detecting the presence of a KRAS mutation by taking the specific KRAS mutation variant into account. Citation Format: Iris van 't Erve, Nina J. Wesdorp, Jamie E. Medina, Leonardo Ferreira, Alessandro Leal, Joost Huiskens, Karen Bolhuis, Jan-Hein T. van Waesberghe, Rutger-Jan Swijnenburg, Daan van den Broek, Victor E. Velculescu, Geert Kazemier, Cornelis J. Punt, Gerrit A. Meijer, Remond J. Fijneman. Clinical impact ofKRASG12, G13, Q61, K117 and A146 mutations in patients with colorectal liver metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 519.

Differential prognostic effect of systemic inflammation in patients with NSCLC treated with immunotherapy or chemotherapy: A post hoc analysis of the phase III OAK trial.

9056 Background: A pro-inflammatory diathesis as measured by the neutrophil-to-lymphocyte ratio (NLR) heralds an adverse disease course in non-small cell lung cancer (NSCLC). Whether the NLR identifies patients who derive a differential degree of benefit from immunotherapy versus chemotherapy is not known. Methods: This post hoc analysis used data from the phase III OAK trial, which randomized previously treated patients with NSCLC to receive atezolizumab or docetaxel. The main objective was to assess the differential impact of pre-treatment NLR on overall survival (OS) depending on the treatment modality. In addition, we assessed patients genomic characteristics according to inflammatory status using circulating free (cf)DNA NGS analysis. Results: A total of 600 and 575 patients with an available NLR were included in the atezolizumab and docetaxel cohort, with a median NLR of 4 (IQR: 2.6-6.7) for the pooled population. NLR ≥ 4 was associated with positive smoking status (88.6% vs 78.1%, p < 0.01), male sex (66.4% vs 57.6%, p = 0.01), worse performance status (71.3% vs 55.2%, p < 0.01), higher number of metastatic sites (63.2% vs 51.6%, p = 0.01), squamous histology (32.1% vs 21.4%, p < 0.01), and tissue KRAS mutation (30% vs 18.7%, p = 0.02), but not with PD-L1 expression, nor with tissue EGFR/ALK status. Pre-treatment NLR of ≥ 4 was more strongly associated with mortality following atezolizumab with an adjusted hazard ratio (HR) of 1.64 (95%CI:1.35-2.01) compared to docetaxel (HR 1.32, 95%CI: 1.08-1.60, multivariable (MVA) interaction p = 0.08. Exclusion of EGFR/ALK positive patients further increased the prognostic ability of baseline NLR in favor of atezolizumab (HR 1.67, 95%CI: 1.35-2.06), as compared with the docetaxel arm (HR 1.24, 95%CI: 1.02-1.52, MVA interaction p = 0.02). The HR for the risk of death for patients with NLR≥ 4/PD-L1 negative tumours (compared to NLR < 4/PD-L1 positive) was significantly higher in the atezolizumab cohort (HR 2.28, 95%CI: 1.72-3.03) than in the docetaxel cohort (HR 1.42, 95%CI: 1.08-1.86, MVA interaction p = 0.01). NGS pretreatment cfDNA data showed that patients with a high blood tumor mutational burden (cut-off 16 Mut/Mb) had a higher median NLR (4.6 vs 3.7, p = 0.01). After adjusting for multiple comparisons, none among the selected variants of interest (EGFR, KRAS, TP53, KEAP1, STK11, SMARCA4, ARID1A and targeted DDR genes), were significantly associated with the NLR. Conclusions: In this post-hoc analysis, a baseline low NLR identifies patients with NSCLC who derive a greater survival benefit from atezolizumab as compared to those identified in the docetaxel cohort, irrespective of genomic features. Patients with a low NLR and PD-L1 positive tumors derive the greatest benefit with immunotherapy and the NLR could complement PD-L1 expression in tailoring treatment in this setting. Clinical trial information: NCT02008227.

KRAS A146 Mutations Are Associated With Distinct Clinical Behavior in Patients With Colorectal Liver Metastases.

PURPOSESomatic KRAS mutations occur in approximately half of the patients with metastatic colorectal cancer (mCRC). Biologic tumor characteristics differ on the basis of the KRAS mutation variant. KRAS mutations are known to influence patient prognosis and are used as predictive biomarker for treatment decisions. This study examined clinical features of patients with mCRC with a somatic mutation in KRAS G12, G13, Q61, K117, or A146.METHODSA total of 419 patients with colorectal cancer with initially unresectable liver-limited metastases, who participated in a multicenter prospective trial, were evaluated for tumor tissue KRAS mutation status. For the subgroup of patients who carried a KRAS mutation and were treated with bevacizumab and doublet or triplet chemotherapy (N = 156), pretreatment circulating tumor DNA levels were analyzed, and total tumor volume (TTV) was quantified on the pretreatment computed tomography images.RESULTSMost patients carried a KRAS G12 mutation (N = 112), followed by mutations in G13 (N = 15), A146 (N = 12), Q61 (N = 9), and K117 (N = 5). High plasma circulating tumor DNA levels were observed for patients carrying a KRAS A146 mutation versus those with a KRAS G12 mutation, with median mutant allele frequencies of 48% versus 19%, respectively. Radiologic TTV revealed this difference to be associated with a higher tumor load in patients harboring a KRAS A146 mutation (median TTV 672 cm3 [A146] v 74 cm3 [G12], P = .036). Moreover, KRAS A146 mutation carriers showed inferior overall survival compared with patients with mutations in KRAS G12 (median 10.7 v 26.4 months; hazard ratio = 2.5; P = .003).CONCLUSIONPatients with mCRC with a KRAS A146 mutation represent a distinct molecular subgroup of patients with higher tumor burden and worse clinical outcomes, who might benefit from more intensive treatments. These results highlight the importance of testing colorectal cancer for all KRAS mutations in routine clinical care.

Targeted Transcriptome and KRAS Mutation Analysis Improve the Diagnostic Performance of EUS-FNA Biopsies in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with a poor prognosis, and current diagnostic tests have suboptimal sensitivity. Incorporating standard cytology with targeted transcriptomic and mutation analysis may improve upon the accuracy of diagnostic biopsies, thus reducing the burden of repeat procedures and delays to treatment initiation. We reviewed the accuracy of 308 endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) diagnostic PDAC biopsies using a large multicenter clinical and biospecimen database, then performed RNA sequencing on 134 EUS-FNA biopsies spanning all stages of disease. We identified a transcriptomic diagnostic gene signature that was validated using external datasets and 60 further diagnostic EUS-FNAs. KRAS digital droplet PCR (ddPCR) analysis was performed and correlated with signature gene expression. The sensitivity of EUS-FNA cytology in diagnosing solid pancreatic masses in our retrospective cohort of 308 patients was 78.6% (95% confidence interval, 73.2%-83.2%). KRAS mutation analysis and our custom transcriptomic signature significantly improved upon the diagnostic accuracy of standard cytology to 91.3% in external validation sets and 91.6% in our validation cohort (n = 60). Exploratory ddPCR analysis of KRAS-mutant allele fraction (MAF%) correlated closely to signature performance and may represent a novel surrogate marker of tumor cellularity in snap-frozen EUS-FNA biopsies. Our findings support snap-frozen EUS-FNA biopsies as a feasible tissue source for the integrated genomic and transcriptomic analysis of patients presenting with PDAC from all tumor stages, including cases with nondiagnostic cytology. Our transcriptome-derived genetic signature in combination with tissue KRAS mutation analysis significantly improves upon the diagnostic accuracy of current standard procedures, and has potential clinical utility in improving the speed and accuracy of diagnosis for patients presenting with PDAC.

Detection of KRAS G12/G13 Mutations in Cell Free-DNA by Droplet Digital PCR, Offers Prognostic Information for Patients with Advanced Non-Small Cell Lung Cancer.

KRAS mutations are found in approximately one third of non-small cell lung cancer (NSCLC) patients. In this study, we aim to investigate whether KRAS G12/G13 mutant allele fraction (MAF) in cell-free DNA (cfDNA) can provide meaningful prognostic information in NSCLC. Multiplex droplet-digital PCR was used to quantitatively assess KRAS G12/G13 MAF in cfDNA from 114 pre-treated advanced disease NSCLC patients. In 14 patients, changes in KRAS G12/G13 MAF were longitudinally monitored during treatment. Plasma KRAS G12/G13 status was associated with poor patients’ outcome in terms of progression-free survival (PFS) (p < 0.001) and overall survival (OS) (p < 0.001). In multivariate analysis, the detection of plasma KRAS mutations was an independent predictor of adverse PFS (HR = 3.12; p < 0.001) and OS (HR = 2.53; p = 0.002). KRAS G12/G13 MAF at first treatment evaluation (T1) was higher (p = 0.013) among patients experiencing progressive disease compared to those with disease control, and increased KRAS MAF at T1 was associated (p = 0.005) with shorter PFS. On the contrary, no association was observed between tissue KRAS mutation status and patients’ prognosis. Our results show that ddPCR-based detection of KRAS G12/G13 mutations in plasma could serve as an independent biomarker of unfavorable prognosis in NSCLC patients. Changes in KRAS MAF can provide valuable information for monitoring patient outcome during treatment.

Early assessment of KRAS mutation in cfDNA correlates with risk of progression and death in advanced non-small-cell lung cancer

BackgroundLiquid biopsy has the potential to monitor biological effects of treatment. KRAS represents the most commonly mutated oncogene in Caucasian non-small-cell lung cancer (NSCLC). The aim of this study was to explore association of dynamic plasma KRAS genotyping with outcome in advanced NSCLC patients.MethodsAdvanced NSCLC patients were prospectively enrolled. Plasma samples were collected at baseline (T1), after 3 or 4 weeks, according to treatment schedule (T2) and at first radiological restaging (T3). Patients carrying KRAS mutation in tissue were analysed in plasma with droplet digital PCR. Semi-quantitative index of fractional abundance of mutated allele (MAFA) was used.ResultsKRAS-mutated cohort included 58 patients, and overall 73 treatments (N = 39 chemotherapy and N = 34 immune checkpoint inhibitors) were followed with longitudinal liquid biopsy. Sensitivity of KRAS detection in plasma at baseline was 48.3% (95% confidence interval (CI): 35.0–61.8). KRAS mutation at T2 was associated with increased probability of experiencing progressive disease as best radiological response (adjusted odds ratio: 7.3; 95% CI: 2.1–25.0, p = 0.0016). Increased MAFA (T1–T2) predicted shorter progression-free survival (adjusted hazard ratio (HR): 2.1; 95% CI: 1.2–3.8, p = 0.0142) and overall survival (adjusted HR: 3.2; 95% CI: 1.2–8.4, p = 0.0168).ConclusionsLongitudinal analysis of plasma KRAS mutations correlated with outcome: its early assessment during treatment has great potentialities for monitoring treatment outcome in NSCLC patients.

Clinical significance of monitoring KRAS in tissue and plasma of pancreatic cancer patients.

745 Background: KRAS monitoringprovides valuable information for early diagnosis and prediction of treatment outcome in colorectal cancer. KRASmutation is observed in only half of colon cancer patients, whereas it is detected in 90% of pancreatic cancer patients. Therefore, investigating tumor DNA in plasma by KRAS monitoringmay be even more valuablein pancreatic cancerpatients. In this study, we elucidated the clinicalsignificance of KRASmonitoring in pancreatic cancer patients during treatment. Methods: KRASin tumor tissues was analyzed for mutations by Scorpion ARMS or RASKET in 83 patients with pancreatic tumors. KRASin plasma was analyzed for mutations(G12D, G12V, G12C, G12A, G12S, G12R, G13D, Q61L, and Q61H) using droplet digital polymerase chain reaction in 88 patients who underwent the curative surgery (N = 45) or the chemotherapy (N = 33) and who had KRAS mutation in their tissues. Results: KRASmutation in tumor tissues was detected in 74 of 83 patients (89.2%). These 74 patients showed significantly poorer prognosis (MST; 32) than the seven patients without mutation (p = 0.03), whose MST were 193. Monitoring of KRASin plasma revealed KRASmutation in 35 of 88 patients (39.8%). In patients who underwent the chemotherapy (N = 33), 2years OS of patients who detected KRASmutation in plasma (N = 23) was 16.4% and them which not detected it (N = 10) was 53.3% (p = 0.18). But in the curative resection group (N = 45), 3years OS of patients who detected KRASmutation in plasma (N = 12) was 16.7% and them which not detected it (N = 33) was 68.2% (p = 0.00). Conclusions: KRASmutation in tissue and plasma could be a valuable predictive and prognostic biomarker in pancreatic cancer patients.

Longitudinal monitoring of KRAS-mutated circulating tumor DNA enables the prediction of prognosis and therapeutic responses in patients with pancreatic cancer.

Liquid biopsies enable the detection of circulating tumor DNA (ctDNA). However, the clinical significance of KRAS-mutated ctDNA for pancreatic cancer has been inconsistent with respect to its prognostic and predictive potential. A total of 422 blood samples were collected from 78 patients undergoing treatments for localized and metastatic pancreatic ductal adenocarcinoma. KRAS mutation in tissues and KRAS ctDNA levels in plasma were determined by RASKET and droplet digital polymerase chain reaction. Longitudinal monitoring of KRAS ctDNA was performed to assess its significance for predicting recurrence and prognosis and for evaluating therapeutic responses to chemotherapy compared with carbohydrate antigen 19-9 (CA19-9). In 67 tumor tissues, discrepancies in point mutations of KRAS were rarely observed among individual patients, implying that one targeted point mutation of KRAS can be determined in tumor tissues prior to longitudinal blood monitoring. One-time blood assessment of KRAS-mutated ctDNA before surgery or chemotherapy was not clearly associated with recurrence and prognosis. Sequential blood monitoring was performed in 39 patients who underwent surgery for potentially resectable tumors. Increased CA19-9 levels were significantly associated with recurrence, but not prognosis (P<0.001, P = 1.0, respectively), whereas emergence of KRAS ctDNA was significantly associated with prognosis (P<0.001) regardless of recurrence. Furthermore, in 39 patients who did not undergo surgery, detection of KRAS ctDNA was a predictive factor for prognosis (P = 0.005). Multivariate analysis revealed that detection of KRAS ctDNA was the only independent prognostic factor regardless of tumor resection (hazard ratios = 54.5 for patients who underwent surgery and 10.1 for patients who did not undergo surgery; P<0.001 for both). Patients without emergence of KRAS ctDNA within 1 year after surgery showed significantly better prognosis irrespective of recurrence (P<0.001). No detection or disappearance of KRAS ctDNA within 6 months of treatment was significantly correlated with therapeutic responses to first-line chemotherapy (P<0.001). Changes in KRAS status provided critical information for the prediction of therapeutic responses. Our study showed for the first time that detection of KRAS ctDNA levels within a short period enables the prediction of prognosis and therapeutic responses in patients with pancreatic cancer.

Abstract 2276: Performance and cost comparison of circulating tumor DNA detection platforms

Abstract Purpose The field of circulating cell free DNA (cfDNA) testing is quickly developing. Multiple platforms to detect hotspot mutations in ctDNA are available, including Bio-Rad droplet digital PCR (ddPCR), BioCartis Idylla, Roche COBAS z480 and Sysmex BEAMing. These platforms vary in the amount of plasma required, the method of ctDNA isolation, the number of hotspots analyzed, and the costs per sample. These factors can impact the applicability of a specific platform in the analysis of ctDNA. In this study we compared these platforms in terms of sensitivity and total costs per sample. Methods The platform comparison was performed as follows: 1. Plasma from six metastatic colorectal cancer (mCRC) patients with known tissue KRAS mutation status was analyzed according to the manufacturers’ protocols. 2. Sensitivity was tested using six constructed reference samples with seven KRAS hotspot mutations at various total input (10ng, 50ng) and mutant allele frequencies (0%, 0.04%, 0.50%); each in four replicates. 3. Twelve mCRC patients were analyzed using equal amounts of ctDNA for each platform. 4. Total costs per sample were evaluated, including costs for consumables, technician hands-on time, equipment and maintenance. Results 1. In six mCRC samples BEAMing (3ml plasma) detected 5/6 mutations, ddPCR and Idylla (both 1ml plasma) 4/6 mutations and COBAS z480 (2ml plasma) 3/6 mutations. 2. In constructed reference samples ddPCR (65%) and BEAMing (46%) yielded the highest sensitivity. With 10ng input BEAMing and COBAS z480 produced “Too little DNA” errors in 60% and 100% of cases, respectively. 3. In twelve mCRC samples, eight had more than 10ng ctDNA and four had less. All platforms were fully concordant for samples with more than 10ng input. Detection rate across twelve samples: Idylla detected 7/11 detectable mutations, COBAS z480 and BEAMing both detected 5/11, ddPCR detected 4/10. 4. BEAMing has the highest cost per sample (€486-€821) whereas ddPCR has the lowest cost per sample (€39-€298). Conclusions A direct comparison of ctDNA mutation detection platforms is complex and should take into account the differences in input and output specifications of the platforms. Factors such as complexity of analysis (Idylla is a low complexity platform, whereas BEAMing requires more specialized training), total costs (varying from ddPCR to BEAMing), sensitivity (ddPCR and BEAMing yielded the highest sensitivity) and the number of mutations evaluated vary greatly between the platforms. All these factors influence ctDNA analysis and will have to be considered when choosing a specific platform for a specific (clinical) question, or when comparing results between studies. Our data provide insight in the comparative performance of four commercial ctDNA analysis platforms, allowing future users to make an informed decision regarding a platform. Acknowledgement Powered by Health~Holland, Top Sector Life Sciences &amp; Health, grant LSHM16047-H005. Citation Format: Daan C. Vessies, Marjolein J. Greuter, Karlijn L. van Rooijen, Theodora C. Linders, Mirthe Lanfermeijer, Kalpana L. Ramkisoensing, Flore E. Grijseels, Boris van Doorn, Gerrit A. Meijer, Miriam Koopman, Veerle M. Coupé, Geraldine R. Vink, Remond J. Fijneman, Daan van den Broek. Performance and cost comparison of circulating tumor DNA detection platforms [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 2276.

The diagnostic accuracy of circulating free DNA for the detection of KRAS mutation status in colorectal cancer: A meta-analysis.

KRAS mutations have been reported as a reliable biomarker for epidermal growth factor receptor (EGFR) targeted therapy and are also associated with poor prognosis in colorectal cancer (CRC) patients. However, limitations of detecting KRAS mutations in tissues are obvious. KRAS mutations in the peripheral blood can be detected as an alternative to tissue analysis. The objective of this meta‐analysis was to evaluate the diagnostic value of cfDNA (circulating free DNA) compared with tissues and to investigate the prognostic potential of cfDNA KRAS mutations in CRC patients. Searches were performed in PubMed, Embase, and Cochrane Library for published studies. We extracted true‐positive (TP), false‐positive (FP), false‐negative (FN), true‐negative (TN) values, survival rate of CRC patients with mutant and wild‐type KRAS and calculated pooled sensitivity and specificity, positive/negative likelihood ratios [PLRs/NLRs], diagnostic odds ratios [DORs], and corresponding 95% confidence intervals [95% CIs]. We also generated a summary receiver operating characteristic (SROC) curve to evaluate the overall diagnostic potential. Totally, 31 relevant studies were recruited and used for the meta‐analysis on the efficacy of cfDNA testing in detecting KRAS mutations. The pooled sensitivity, specificity, PLR, NLR, and DOR were 0.637 (95% CI: 0.607‐0.666), 0.943 (95% CI: 0.930‐0.954), 10.024 (95% CI: 6.912‐14.535), 0.347 (95% CI: 0.269‐0.447), and 37.882 (95% CI: 22.473‐63.857), respectively. The area under the SROC curve was 0.9392. Together, the results suggest that detecting KRAS mutations in cfDNA has adequate diagnostic efficacy in terms of specificity. There is a promising role for cfDNA in the detection of KRAS mutations in CRC patients. However, prospective studies with larger patient cohorts are still required before definitive conclusions of the prognostic potential of cfDNA KRAS mutations in CRC patients were drawn.

Clinical significance of monitoring KRAS in tissue and plasma of pancreatic cancer patients.

254 Background: KRAS mutation is observed in 90% of pancreatic cancer patients. Therefore, investigating tumor DNA in plasma by KRAS monitoring may be even more valuable in pancreatic cancer patients. Methods: We collected the tissue and plasma of 78 pancreatic cancer patients (surgery group; 39, non-surgery group; 39). KRAS mutation in the tissue and mutated circulating tumor DNA (MctDNA) in plasma was detected by digital polymerase chain reaction in 78 patients. Identical KRAS mutation detected in tissue (ex. 12D, 12V) was monitored in plasma. Results: KRAS mutation in the tissue was detected in 65 of 73 patients. KRAS assessment in the tissue was not performed in 5 patients, because of tissues small amounts of tissue materials by biopsy. These 65 patients with KRAS mutation in tissue showed poorer prognosis (3 years OS; 23.4%) than 8 patients without mutation (3 years; 66.7%). MctDNA in plasma of surgery group was seen in 14 of 39 patients. Thirteen in 14 patients with MctDNA showed recurrence and 12 patients were dead. These 14 patients with MctDNA in plasma showed significantly poorer prognosis (2 years OS; 16.3%) than 25 patients without mutation (3 years; 71.6%) (p = 0.00). Univariate analysis revealed that poor differentiation and the detection of MctDNA were independent factors to predict poor survival in surgery group. The detection of MctDNA was confirmed to be an independent factor in multivariate analysis (Hazard ratio; 31.25). MctDNA in plasma of non-surgery group was seen in 28 of 39 patients. But, MctDNA in 6 patients was disappeared in clinical course. These 6 patients and 11 patients without MctDNA displayed better prognosis (2 years OS; 72.1%) than 22 patients with MctDNA (2 years OS; 12.1%) with significance (p = 0.0001). Univariate and multivariate analysis revealed that no treatment and the detection of MctDNA were independent factors to predict poor survival in non-surgery group. (Hazard ratio; 8.78, 4.76, respectively). Conclusions: KRAS monitoring in plasma reflects tumor dynamics. The appearance of MctDNA during KRAS monitoring provides important information for the treatment of pancreatic cancer patients.

Repeated mutKRAS ctDNA measurements represent a novel and promising tool for early response prediction and therapy monitoring in advanced pancreatic cancer

BackgroundThe presence of mutated KRAS (mutKRAS ctDNA) in plasma samples has been consistently shown to be a negative prognostic indicator in pancreatic cancer (PC). Only small pilot studies have evaluated the value of serial mutKRAS ctDNA-measurements in PC. Patients and methodsThe aim of the present study was to explore the potential of repeated mutKRAS ctDNA measurements for response prediction and therapy monitoring in advanced PC patients. We used the BEAMing technology to determine levels of mutKRAS ctDNA, CA 19-9, CEA and CYFRA 21-1 in 284 plasma samples of 54 patients with advanced PC receiving gemcitabine-based chemotherapy. Absolute levels and kinetics of mutKRAS ctDNA, CA 19-9, CEA and CYFRA 21-1 were correlated to radiological response, progression-free and overall survival. ResultsmutKRAS ctDNA was present in a majority of advanced PC patients (n = 36/54, 67%) and indicated tissue KRAS mutation status with a high sensitivity (75%) and specificity (100%). The presence of mutKRAS ctDNA, as well as higher levels of CA 19-9, CEA and CYFRA 21-1 before initiation of the first-line chemotherapy, was significantly correlated to an adverse overall survival. During therapy, changes in mutKRAS ctDNA levels were more rapid and pronounced than changes in protein-based tumor markers. A decrease in mutKRAS ctDNA levels during therapy was an early indicator of response to therapy, while there was no significant correlation between kinetics of CA 19-9, CEA or CYFRA 21-1 and response to chemotherapy during the first four weeks of treatment. Repeated mutKRAS ctDNA measurements during follow-up appeared to be superior to protein-based tumor markers in detecting progressive disease (sensitivity: 83%, specificity: 100%). ConclusionmutKRAS ctDNA kinetics appear to be a powerful and highly specific tool in early response prediction and therapy monitoring of advanced PC patients receiving chemotherapy.

Plasma ctDNA RAS/RAF mutations analysis for monitoring overall survival (OS) and heterogeneity in metastatic colorectal cancer patients (mCRC).

701 Background: The study was designed to estimate the clinical usefulness of genetic analysis for circulating tumor DNA (ctDNA) by digital polymerase chain reaction in patients with mCRC. Methods: We compared B-RAF and K/N/H-RAS mutations detected in surgical or endoscopic ultrasound-guided fine-needle aspiration biopsy tissue DNA and in ctDNA from 63 patients with mCRC. p53 mutational status and PD-L1 expression in the serum and in tissues respectively (positivity H score &gt; 55) were also analyzed with a comparison with survival rates. Results: The frequencies of K-RAS mutations in tissue samples at G12V, G12D, and G12R in codon 12 were 18 of 63 samples (28.57%), 5 of 63 samples (7.93%), and 6 of 63 samples (9.52%), respectively. Conversely, the rates of the mutations in ctDNA were 22 of 63 samples (34.92%), 6 of 63 samples (9.52%), and 9 of 63 samples (14.28%), respectively. No H-RAS or N-RAS mutation were detected. 2 tissue samples harbored a BRAF mutation (3.17%) in a complete discordance with same mutation detected in 6 different serum ctDNA (9.52%). Overall, the K-RAS mutation rates in tissue and ctDNA were 45.72% and 58.72%, respectively, and the concordance rate between them was 29 of 37 samples (78,37%). OS did not appear to differ by the presence of K-RAS mutations in tissue or in the serum DNA, but the OS of patients with B-RAF mutations in ctDNA was significantly shorter than that of patients with tissue K-RAS/B-RAF mutations (11 vs 19 months, p &lt; 0.0001). PD-L1 positivity was significantly associated with p53 mutational status (concordance: 82%) and with a worse outcome for OS. Conclusions: Analysis of ctDNA is a new useful procedure for detecting mutations in mCRC patients. This non invasive method may have great potential to understand tumor heterogeneity and appear a new strategy for the planning therapies as well as for predicting survival.

K-ras mutation detection using a novel amplification refractory mutation system-based quantitative polymerase chain reaction with locked nucleic acid blocker in plasma among colorectal cancer patients

Objective To determine whether plasma circullating cell-free DNA (cfDNA) is a viable alternative approach for K-ras mutation testing in colorectal cancer (CRC) patients. Methods The study included 155 CRC patients. An a novel amplification refractory mutation system-based quantitative polymerase chain reaction with locked nucleic acid (LNA-ARMS-PCR) was developed to assess the K-ras mutation in preoperative plasma and paired tumor tissues. Firstly intra-tumoral heterogeneity analysis was used to determine whether it was a robust mutation detection method for the detection of low-abundance K-ras mutations. Results The newly built LNA-ARMS-PCR was a robust mutation detection method to escape the most false negative caused by intra-tumoral heterogeneity. The positive consensus rate and negative consensus rate was 35.3% and 100.0%, between plasma and tumor tissue K-ras mutation detection, respectively. The majority of K-ras mutations detected in tumors were also found in plasma(15/18 (83.3%)) in patients of stage Ⅳ. The accordance of plasma and tumor mutation are strongly correlated to TNM stage and cfDNA level. Conclusion K-ras analysis in plasma cfDNA is only a viable alternative to tissue analysis in mCRC (stage Ⅳ). Quantitative levels of cfDNA and plasma mutation positivity are strongly correlated. Key words: Colorectal cancer; K-ras; Locked nucleic acid; Amplification refractory mutation system

Abstract 5237: Monitoring minimal residual disease by urinary or plasma circulating tumor DNA of KRAS mutation burden in colorectal cancer patients with resectable liver metastases

Abstract Background: Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the third leading cause of cancer deaths. Over half of patients with CRC will develop liver metastases. Surgical resection, in combination with systemic therapies, greatly improves long-term outcomes, and around 40% of patients with resected liver limited disease are alive 5 years after diagnosis. While tumor staging and radicality of surgery are commonly used for prognostic assessment, better non-invasive markers are needed for monitoring chemo-responsiveness, following minimal residual disease (MRD), and guiding complex treatment decisions in these patients. This study evaluated the utility of quantitating KRAS mutation burden in urinary and plasma ctDNA as a means of monitoring MRD in surgical CRC patients with liver limited metastases. Methods: We developed a novel, NGS-based method for enrichment and quantitative detection of KRAS mutations in urinary and plasma ctDNA. A blinded retrospective study was conducted on 20 Stage I-IV CRC patients, 15 of whom had undergone curative or palliative intent surgical resection of primary tumor or liver metastases in combination with various systemic therapies. Results: Archived, matched urine and plasma samples were collected from 20 patients with KRAS positive primary tumor. For operable patients, specimens were collected prior to surgery, during and immediately after surgery, plus additional time points post-surgery. A total of 193 plasma and urine samples archived for 3-5 years were tested. All 101 of 101 plasma samples (100%) and 79 of 92 urine samples (86%) had sufficient DNA and were deemed evaluable. In a blinded analysis, a correct KRAS mutation that correlated with KRAS mutation in tissue was identified in 95% of evaluable baseline plasmas (19 of 20) and 92% of evaluable baseline urines (11 of 12). In one patient, a KRAS mutation distinct from that identified in the tumor biopsy was detected consistently across all serial ctDNA samples. Overall, we observed a clear correlation and highly comparable fold change between plasma and urinary ctDNA KRAS levels on treatment. Significantly, in all patients with curative intent surgery, ctDNA KRAS levels were undetectable in urine or plasma after surgery. In contrast, in 8 of 10 patients with incomplete, palliative surgery, the ctDNA KRAS signal remained detectable or increased after surgery. Further correlation between ctDNA KRAS and clinical outcomes will be discussed. Conclusion: We demonstrate for the first time that quantitative changes of mutational KRAS burden in plasma and urinary ctDNA are highly correlated. We further demonstrate clinical applicability of urinary ctDNA KRAS analysis for monitoring quantitatively, with single molecule sensitivity, the MRD post-surgery for CRC patients with liver resectable metastases. Supported by Grant no. 13660. Citation Format: Vlada Melnikova, Jason C. Poole, Cecile Rose T. Vibat, Lucie Benesova, Barbora Belsanova, Saege Hancock, Latifa Hassaine, Errin Samuelsz, Timothy T. Lu, Mark G. Erlander, Marek Minarik. Monitoring minimal residual disease by urinary or plasma circulating tumor DNA of KRAS mutation burden in colorectal cancer patients with resectable liver metastases. [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 5237. doi:10.1158/1538-7445.AM2015-5237

Abstract A22: Sensitive detection of mutant KRAS in colorectal tissue using the multiplex QuARTS™ assay

Abstract Background: Colorectal cancer (CRC) is the second leading cause of cancer deaths in the United States, yet it is potentially the most treatable and preventable cancer with effective screening. Exact Sciences has developed a multi-biomarker CRC screening test that targets DNA methylation, DNA mutations, and hemoglobin in stool. The aim of this study was to evaluate the analytical accuracy of a novel assay for the detection of KRAS mutation sequences at Codons 12 and 13 in well-characterized colorectal tissues. Methodology: A multiplexed KRAS assay was designed utilizing QuARTS (Quantitative Allele-specific Real-time Target and Signal amplification), a highly sensitive technology that combines allele-specific DNA amplification with invasive cleavage chemistry to generate signal during each amplification cycle similar to real-time PCR. The assay, which detects seven KRAS mutations and the reference gene β-actin, was used to assess 87 colorectal tissue samples (52 CRCs, 16 adenomas ≥ 1cm, and 19 normal epithelia) as determined by Mayo Clinic Pathology. Samples were obtained by microdissection of fresh frozen tissue biopsies. DNA was extracted by Mayo Clinic using a standardized chloroform/phenol methodology. The genotypes of each sample were established using dye terminator dideoxy sequencing in both the forward and reverse orientations. Copy numbers of KRAS mutations and β-actin were determined by conventional comparison against standard curves. KRAS data are reported as percent mutation and calculated by dividing mutant copies by β-actin copies and multiplying by 100. Results: Based on sequencing data: the 52 CRC samples contained 22 KRAS mutations and 30 wild-type genotypes, the 16 adenomas ≥ 1cm contained 8 mutations and 8 wild-type genotypes, and the 19 normal tissues contained all wild-type genotypes. The QuARTS assay detected 100% of the KRAS mutations in the CRC and adenomas and provided excellent differentiation between wild-type and mutation, with the highest percent KRAS mutation of normal wild-type samples at 0.55% and the lowest percent mutation of KRAS positive samples at 8.34%. Based on this data, this assay has the potential to be more sensitive analytically than standard sequencing. Conclusion: The QuARTS assay detects KRAS mutations in tissue with high sensitivity and specificity. This assay method could provide an accurate and high throughput approach to detection of KRAS mutations in biospecimens for multiple clinical applications. Citation Information: Cancer Prev Res 2011;4(10 Suppl):A22.

Low Correspondence Between K-ras Mutations in Pancreatic Cancer Tissue and Detection of K-ras Mutations in Circulating DNA

K-ras is the most frequently mutated gene in pancreatic cancer; reported rates range from 70% to 90%. The aim of this study was to evaluate the correspondence between K-ras mutations in pancreatic cancer tissue and in circulating DNA and the value of K-ras mutations as serological marker. The research was conducted in 30 patients with pancreatic cancer in whom both plasma and neoplastic tissues were available. Such research was extended to circulating DNA isolated from 40 patients with chronic pancreatitis. Mutations in codon 12 were examined by mutant allele-specific amplification method and by direct sequencing. Serum values of routinely used tumor markers such as carbohydrate antigen (Ca) 19.9, carcinoembryonic antigen, Ca 50, and Ca 242 have been tested in all the patients enrolled in this study. K-ras mutations were detected in 70% of neoplastic tissue samples, but no mutated DNA resulted in circulating DNA samples. The 60% of patients with tissue K-ras mutation showed elevation of some tumor markers among Ca 19.9, carcinoembryonic antigen, Ca 50, and Ca 242. As a whole, these last showed low sensitivity (20%-56.67%) and specificity (56.67%-77.5%) when compared with chronic pancreatitis. Over the years, there has been no change in the direction of an earlier diagnosis by serological markers, and also, these data indicate that K-ras mutation in serum is an unsatisfactory method for the detection in patients with pancreatic cancer as well as in patients with high risk of progression toward neoplastic pancreatic disease.