Detectable KRAS Research Articles

Abstract 1144: Dynamic changes in circulating tumor DNA (ctDNA) in patients treated with sotorasib for KRAS G12C mutant non-small cell lung cancer

Abstract Background: Sotorasib was recently approved for KRAS G12C mutant advanced non-small cell lung cancer (NSCLC). We explored whether the presence of specific mutations and dynamic changes in circulating tumor DNA (ctDNA) would be of prognostic/predictive value for patients (pts) with KRAS G12C mutations on sotorasib. Methods: Serial plasma was collected from pts with advanced KRAS G12C mutant NSCLC treated on the registrational CodeBreaK 100 trial, including at baseline (cycle 1, day 1, C1D1), cycle 2, day 1 (C2D1), and at end of treatment (EOT). At one site (Memorial Sloan Kettering), plasma was collected at additional timepoints, including C1D2 and C1D8. Plasma was analyzed using the Resolution Bioscience ctDX LungTM assay, a 23-gene panel. Results: Plasma was collected from 179 pts, for a total of 624 evaluable samples. At baseline, 77% of pts (129/168) had detectable plasma KRAS G12C mutations. Lower G12C allele frequency (AF) at baseline correlated with better % change in radiographic Sum of Diameters (SoD) (Spearman’s rho=0.22, p <0.001). Pts without baseline detectable plasma KRAS G12C had a median PFS of 11.5 months as compared to 4.1 months in those with detectable G12C (HR 2.8, p<0.001), and were more likely to respond (HR = 2.76, p <0.001). Pts with no baseline detectable ctDNA also had longer PFS (11.1 versus 5.1 months, HR 2.07, p=0.01). While responses were seen across a variety of genomic alterations, complete responders had fewer co-mutations overall. 75% (3) of complete responders (CRs) had no mutations of any kind detected vs 12% (19) of non-CRs (p=0.008, Fisher exact test). The number of altered genes was lower in responders (p<0.001, Poisson regression), and CRs alone tended to have fewer altered genes (p=0.06), though the number of cases was very small (4). EGFR amplification at baseline was observed in patients who achieved a partial response (PR), but not a CR. The early kinetics of ctDNA comparing C1D2 to C1D1 and C2D1 to C1D8 did not differ between pts who had a decrease in SoD by RECIST on first scans (completed around C3D1) and those who had an increase (p>0.1, tested by within and between group medians); both increases and decreases in G12C AF were seen at these time points regardless of radiographic changes. A subset of both responders (26% [13/50]) and non-responders (54% [42/78]), had a transient drop in ctDNA between C1D1 and C2D1 with a subsequent increase after C2D1. Those with no detectable ctDNA at C2D1 had longer median PFS (8.3 months) compared to those with detectable ctDNA (4.4 months, p=0.007). Conclusion: At baseline, lower G12C AF and lower number of altered genes were associated with improved outcomes, suggesting there may be benefit to early initiation of sotorasib when co-mutational burden is low. Changes in ctDNA early in treatment are dynamic. Absence of detectable ctDNA at C2D1 may be associated with longer PFS. Citation Format: Yonina R. Murciano-Goroff, Ferdinandos Skoulidis, Vamsidhar Velcheti, Gerald S. Falchook, Grace Dy, Suresh Ramalingam, Martin J. Edelman, Jürgen Wolf, Antoine Italiano, Jorge S. Reis-Filho, Pedram Razavi, Alexander Drilon, Sandra Misale, Rona Yaeger, Mark Li, Antreas Hindoyan, Agnes Ang, Abraham A. Anderson, David Hong, Piro Lito, Ramaswamy Govindan, Bob T. Li. Dynamic changes in circulating tumor DNA (ctDNA) in patients treated with sotorasib for KRAS G12C mutant non-small cell lung cancer [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 1144.

Abstract 2137: Clinical utility of circulating tumor DNA in patients with advanced KRAS G12C-mutated NSCLC treated with sotorasib

Abstract Introduction: The CodeBreaK 200 trial showed that in patients (pts) with advanced KRAS G12C mutated (KRAS+) NSCLC sotorasib, a KRAS G12C-specific inhibitor, is superior to docetaxel for progression free survival (PFS) (HR 0.66) with a one-year PFS of 25%. We hypothesized that the detection of circulating tumor DNA (ctDNA) in plasma could allow for treatment response prediction and longitudinal monitoring. We analyzed serial plasma samples at baseline and within 3 months of start of sotorasib to evaluate ctDNA changes and correlation with clinical response. Methods: Pts with sotorasib treated KRAS+ NSCLC were prospectively enrolled in our biomarker START-TKI study (NCT05221372) after written informed consent. Plasma samples were collected prior to treatment (T0) and at first response evaluation (T1). The TruSight Oncology 500 ctDNA panel was used for mutation detection in cell-free DNA (cfDNA). cfDNA KRAS/TP53/STK11/KEAP1 status was determined and compared to tumor tissue pathology reports to filter out false positives. Radiological response and PFS was assessed per RECIST 1.1. Results: Between May 2021 and August 2022, 35 pts were included (table 1). Of these, 29 (83%) had detectable ctDNA KRAS G12C at T0, and 24 had both T0 and T1 samples. A decrease in variant allele frequency (VAF) at T1 compared to T0 was observed in 88% (n=21); 42% (n=10) showed complete clearance of ctDNA KRAS G12C. Six-months PFS was 83% in T0 negative pts (n=6) versus 43% in T0 positive pts (n=29); and 65% in pts with complete clearance (n=10) versus 14% in pts with incomplete clearance (n=11). VAF increase was seen in 3 pts, of which 1 had progression at T1. Conclusions: In pts with KRAS+ NSCLC treated with sotorasib, baseline ctDNA and ctDNA clearance within 3 months of treatment correlated with treatment response. Pts with undetectable KRAS ctDNA at baseline, or with complete clearance at first evaluation, had superior PFS. PFS will be updated as follow-up duration extends. Table 1. Patient cohort Liver metastasis KRAS p.G12C ctDNA at T0, median VAF % (range) KRAS p.G12C ctDNA at T1, median VAF % (range) Median change in VAF % (range) TP53/STK11/KEAP1 ctDNA T0 T1 response Reason EOT ꝉ Time on treatment (months) * Negative at T0 (n=6) No = 6 0 NE NE NE 1x PR; 4x SD; 1x PD 2x PD; 4x ongoing >8 months 6 (1 - 11) Positive at T0 (n=29) No = 20; Yes = 9 2.6 (0.1 - 46.7) 0.6 (0.1 - 38.9) -95% (-100 - +39) 4x TP53; 2x STK11; 1x STK11●; 2x KEAP1; 2x KEAP1●; 2x TP53 + KEAP1; 1x TP53 + STK11● 4x PR; 21x SD; 2x PD; 2x NE 19x PD; 3x toxicity; 6x ongoing >4 months; 1x loss to follow up 3 (1 - 15) Complete clearance at T1 (n=10) No = 6; Yes = 4 1.4 (0.1 - 8.4) 0 100% 2x TP53; 1x TP53 + KEAP1; 1x KEAP1; 1x KEAP1● 1x PR; 9x SD 6x PD; 1x toxicity; 2x ongoing >6 months; 1x loss to follow up 5 (1 - 11) Incomplete clearance at T1 (n=11) No = 7; Yes = 4 3.6 (1.3 - 46.7) 0.4 (0.1 - 29.4) -89% (-23 - -99) 2x TP53; 1x STK11; 1x STK11●; 2x TP53 + STK11●; 1x TP53 + STK11 + KEAP1; 1x STK11 + KEAP1 3x PR; 7x SD; 1x PD 8x PD; 2x toxicity; 1x ongoing >4 months 3 (1 - 8) Increase in VAF at T1 (n=3) No = 2; Yes = 1 1.0 (0.5 - 35.2) 1.1 (0.6 - 38.9) +11% (+10 - +39) 1x TP53●; 1x TP53 + KEAP1; 1x STK11● + KEAP1● 1x PD; 2x SD 3x PD 3 (1 - 15) Positive T0, no T1 available (n=5) No = 5 6.2 (0.3 - 24.6) NE NE 1x TP53; 1x TP53●; 1x KEAP1; 1x STK11 + KEAP1 3x SD; 2x NE 2x PD; 3x ongoing >3 months 2 (1 - 3) NE = Not evaluated; ● = Positive cell-free DNA (cfDNA), not tested in tumor tissue; PR = Partial response; SD = Stable disease; PD = Progressive disease; EOT = End of treatment; ꝉ for PFS analysis patients who discontinued treatment due to toxicity were censored at treatment discontinuation, and patients with ongoing sotorasib treatment were censored at data cut off (16-Nov-2022); * = excluding ongoing patients. Citation Format: Sophie M. Ernst, Ronald van Marion, Peggy N. Atmodimedjo, Evert de Jonge, Ron H. Mathijssen, Marthe S. Paats, Peter de Bruijn, Ron H.N. van Schaik, Hendrikus J. Dubbink, Anne-Marie C. Dingemans. Clinical utility of circulating tumor DNA in patients with advanced KRAS G12C-mutated NSCLC treated with sotorasib [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 2137.

The identification of reversion mutations in patients with advanced pancreatic cancer and germline or somatic BRCA or PALB2 variants who were treated with maintenance rucaparib.

734 Background: Maintenance PARP inhibition (PARPi) extends progression-free survival and improves quality of life for patients (pts) with advanced, platinum-sensitive pancreatic cancer (PC) and BRCA or PALB2 variants. However, most will experience progression. PARPi resistance mechanisms are poorly defined in PC. Cell-free (cf)DNA analysis can detect some known classes of resistance mechanisms, like reversion mutations, and other potentially prognostic and predictive genomic features. Methods: Pts with advanced, platinum-sensitive pancreatic cancer and pathogenic germline or somatic BRCA1, BRCA2, or PALB2 variants were treated with maintenance rucaparib on clinical trial. cfDNA was collected at baseline and progression and analyzed with the GuardantOMNI 500-gene liquid biopsy. Time to event analysis was performed from index date of enrollment until endpoint (PFS, OS, and PFS2). Associations were tested by the log-rank test with adjustment. Results: The trial enrolled 42 pts, of whom 31 have progressed. cfDNA was available for 41 pts at baseline and 30 pts at progression; 88% had baseline detectable cfDNA. Two pts had baseline reversion mutations, 5 had new reversion mutations at progression. Of 21 pts who had tissue NGS, 17 pts had a KRAS variant in the tumor, 12 of whom had detectable cfDNA at either baseline or progression. Of the 41 patients with cfDNA samples, 10 pts had baseline KRAS mutations detected in plasma; an additional 10 pts had a detectable plasma KRAS mutation at progression. Outcomes are shown. Of those who had progressed, pts with acquired reversion mutations had shorter OS (p<0.001) and PFS (p = 0.018) on rucaparib than those without reversion mutations. Of those who received chemo after progression (n=23), PFS2 was shorter for pts with acquired reversions compared to those with no reversions (p = 0.038). KRAS mutation detection at baseline was observed with higher overall somatic allele fraction in cfDNA and a trend toward shorter PFS and OS. Conclusions: Acquired reversion mutations were infrequent but associated with worse outcomes. Other causes of resistance may be dominant. Detection of KRAS mutation in the peripheral blood may be associated with disease burden and clinical outcome. [Table: see text]

Evaluation of circulating cell-free KRAS mutational status as a molecular monitoring tool in patients with pancreatic cancer

BackgroundPancreatic carcinoma carries a devastating prognosis and is the 4th leading cause for cancer related death in the US and most European countries. Apart from imaging and CA 19-9, pancreatic carcinoma is still lacking reliable markers to assess tumor dynamics and to monitor treatment response over time. The aim of this study was to evaluate the feasibility of cell free tumor-DNA (cft-DNA), respectively KRAS mutation in peripheral blood, detection as a prognostic and predictive value for chemotherapy monitoring. MethodsSerial plasma samples from 42 patients with KRAS mutated pancreatic cancer were prospectively collected and the ctKRAS Mutation Assay (Idylla™, Biocartis, Mechelen, Belgium) of cft-DNA was performed on 29 patients that did not receive curative surgery and went on to palliative chemotherapy. To monitor cft-DNA KRAS mutation levels during treatment quantitative assessment of cft-DNA was performed at baseline and during follow up at predetermined times. ResultsAll 29 patients included in our analyses had a detected KRAS mutation in the tumor biopsy. In almost half (48.2%) of patients a KRAS mutation could also be detected in peripheral plasma. Patients with detectable KRAS mutations before treatment start in plasma had a significantly worse survival (16.8 months vs not reached, p < 0.031 and HR 3.303). Looking for a dynamic assessment of tumor response, we found a statistically significant association between the KRAS mutant ratio from first staging CT scan to basal levels with tumor response or progress (p = 0.014). ConclusionPerforming KRAS testing from peripheral blood for patients, who have no elevated tumor markers, might be a novel option for treatment monitoring complementing routine imaging techniques.

Abstract 867: Incidence of molecular alterations in KRAS and other known cancer genes in patients with pancreatic cancer assessed with a commercial genomic profiling panel compared to TCGA results

Abstract Background: The Cancer Genome Atlas (TCGA) characterizes somatic mutations of pancreatic cancer (PC) patients (pts) using whole-exome sequencing (WES, mean coverage 405x) of fresh frozen tumor tissues and matched blood samples. In addition, it includes a confirmation of the key genomic findings by a targeted sequencing panel (~644x) and microfluidic PCR-based sequencing (~30,000x). For the vast majority of patients who do not undergo resection, however genomic characterization of formalin-fixed, paraffin-embedded (FFPE) biopsy samples is the only available assessment. In order to assess this NGS testing process at Baylor College of Medicine, we sought to confirm that routinely collected diagnostic FFPE specimens could be used for sequencing without compromising the detection rates of key gene alterations in PC pts. Methods: Matched tumor and normal specimens (peripheral blood or saliva) from 59 Baylor College of Medicine PC pts were sequenced using Tempus|xT (Tempus Labs, Chicago, IL), a 648-gene DNA sequencing panel (avg. depth of 500x) integrated with whole-transcriptome RNA sequencing. Retrospective analysis of detection rates of all clinically reported pathogenic mutations was performed using LENS, a proprietary application that allows interrogation of large de-identified molecular and clinical datasets. Results: The tumor cellularity of the 59 FFPE samples as assessed by board-certified pathologists ranged from 10% to 80%. Approximately 88% (52/59) of these samples had detectable KRAS alterations, relative to 93% identified in the TCGA database. KRAS G12D, G12V, and G12R were the most frequent findings (29/59, 11/59, and 8/59, respectively). The average KRAS variant allele fraction (VAF) was 16.87% (range 1.7%- 42.2%). Alterations in CDKN2A were detected with a noticeably higher incidence as compared to the TCGA database, 37.2% (22/59) vs 17.4%, respectively. Conversely, TP53 and SMAD4 had similar detection incidence between the two platforms. In addition, 13.5% (8/59) of PC pts had a somatic or germline mutation in the homologous recombination (HR) pathway, with 4 pts having a BRCA2 alteration (6.7%). Finally, one patient was tumor mutational burden (TMB)-High and microsatellite instability (MSI)-High, both of which are tumor-agnostic indications for checkpoint inhibition. Conclusions: An analysis of PC FFPE tissues using the Tempus|xT panel at a clinical depth of 500x revealed a detection rate of KRAS, TP53, and SMAD4 alterations comparable to the TCGA database. Citation Format: Alexander D. Le, Bing Zhang, Kim C. Worley, David Wheeler, Benjamin L. Musher, Heidi Dowst, Amy Wood, Jeff Schaeffer, Nick Olsen, Lorenzo Grego, Matt Sonnleitner, William E. Fisher. Incidence of molecular alterations in KRAS and other known cancer genes in patients with pancreatic cancer assessed with a commercial genomic profiling panel compared to TCGA results [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 867.

Dynamic Regulation of Expression of KRAS and Its Effectors Determines the Ability to Initiate Tumorigenesis in Pancreatic Acinar Cells.

Pancreatic acinar cells are a cell type of origin for pancreatic cancer that become progressively less sensitive to tumorigenesis induced by oncogenic Kras mutations after birth. This sensitivity is increased when Kras mutations are combined with pancreatitis. Molecular mechanisms underlying these observations are still largely unknown. To identify these mechanisms, we generated the first CRISPR-edited mouse models that enable detection of wild-type and mutant KRAS proteins in vivo. Analysis of these mouse models revealed that more than 75% of adult acinar cells are devoid of detectable KRAS protein. In the 25% of acinar cells expressing KRAS protein, transcriptomic analysis highlighted a slight upregulation of the RAS and MAPK pathways. However, at the protein level, only marginal pancreatic expression of essential KRAS effectors, including C-RAF, was observed. The expression of KRAS and its effectors gradually decreased after birth. The low sensitivity of adult acinar cells to Kras mutations resulted from low expression of KRAS and its effectors and the subsequent lack of activation of RAS/MAPK pathways. Pancreatitis triggered expression of KRAS and its effectors as well as subsequent activation of downstream signaling; this induction required the activity of EGFR. Finally, expression of C-RAF in adult pancreas was required for pancreatic tumorigenesis. In conclusion, our study reveals that control of the expression of KRAS and its effectors regulates the sensitivity of acinar cells to transformation by oncogenic Kras mutations. SIGNIFICANCE: This study generates new mouse models to study regulation of KRAS during pancreatic tumorigenesis and highlights a novel mechanism through which pancreatitis sensitizes acinar cells to Kras mutations.

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.

Abstract 709: The utility of plasma ctDNA for detection of KRAS G12C and other mutations in lung cancers

Abstract Background: KRAS is the most common oncogene in lung cancer, but has historically been considered undruggable. The recent development of mutant-selective KRAS G12C inhibitors has for the first time created potential therapeutic opportunities for this unmet need. Simultaneously, circulating tumor DNA (ctDNA) is increasingly being used to detect targetable oncogenes in patients with metastatic lung cancers. There is limited data regarding the utility of plasma ctDNA in specifically identifying KRAS G12C mutations. Methods: Plasma was collected from 599 patients with lung cancer seen at Memorial Sloan Kettering Cancer Center between 10/2016 and 1/2019. ctDNA sequencing was performed using the ctDX-Lung Assay (Resolution Bioscience; Kirkland, WA). Tissue DNA sequencing was carried out using the MSK-IMPACT assay. Results: Mutations in KRAS (KRAS+) were detected in 129 patients (21.5%). Of patients with KRAS+ lung cancers, 116 had metastatic disease at the time of plasma testing. Plasma testing was carried out within 90 days of metastatic diagnosis in 92 of the 116 metastatic KRAS+ patients (79.3%), of whom 66 patients had both plasma and tissue sequencing available for comparison. 59 of the 66 patients (89.4%) had not had systemic treatment at the time of plasma testing. Average turn-around-time (TAT) for ctDNA testing in this cohort of 66 patients was 10 days, while average TAT for tissue sequencing was 22 days. ctDNA detected a KRAS mutation in 48 of the 66 patients (72.7%). A G12C mutation was found in the tissue and/or blood from 29 patients (43.9%), while 37 patients (56.1%) had other KRAS mutations. In one patient with a G12C mutation detected on plasma testing, no KRAS mutation or other known oncogenic drivers were found on tissue testing, though with limited tumor cells in the tissue sample. 75.6% of the patients with G12C mutations had KRAS detectable in the plasma, as compared to 70.3% of patients with other KRAS mutations (p=0.6). Within the cohort of patients with paired tissue and plasma testing, at a median follow-up of 202 days post-metastatic diagnosis, survival was longer in those patients without detectable KRAS in the plasma as compared to those patients with detectable plasma KRAS (log rank (Mantel-Cox), p&amp;lt;0.001, log rank HR 7.9, 95% CI: 3.7-16.8). Conclusion: Plasma testing was able to rapidly detect KRAS G12C mutations in the majority of patients with this alteration, which may guide G12C inhibitor therapy. In our cohort, patients that shed KRAS mutant DNA into the plasma had shorter survival. Citation Format: Yonina R. Murciano-Goroff, Kathryn C. Arbour, Michael D. Offin, Hai-Yan Y. Tu, Emily S. Lebow, Tristan S. Shaffer, Caterina Bertucci, Syed A. Hosseini, Kavita Garg, Lee P. Lim, Mark Li, Jason C. Chang, Jorge S. Reis-Filho, Pedram Razavi, James M. Isbell, Gregory J. Riely, David M. Hyman, Piro Lito, Bob T. Li. The utility of plasma ctDNA for detection of KRAS G12C and other mutations in lung cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 709.

Evaluation of circulating tumor DNA as a biomarker in pancreatic cancer with liver metastasis.

Pancreatic cancer is an aggressive, solid tumor, with a grave prognosis. Despite surgical treatment in patients with pancreatic cancer, the rate of recurrence is high. In addition, although tumor biomarkers are frequently used to confirm advanced pancreatic cancer, this is not accurate and the biomarkers currently used cannot indicate prognosis. This study sought to evaluate circulating tumor DNA as a tumor biomarker to prognosticate pancreatic cancer. Patients with advanced pancreatic cancer and liver metastasis (N = 104) were included, and blood samples were collected from all patients. The mutant allele frequency was measured using amplicon-based deep sequencing on a cell-free DNA panel covering 14 genes with > 240 hot spots. In patients with advanced pancreatic cancer, 50% (N = 52) had detectable ctDNA levels, with TP53 (45%, N = 47) and KRAS (42.3%, N = 44) mutations the most common. Patients with detectable circulating tumor DNA levels also had significantly worse overall survival and progression free survival than ctDNA negative patients (8.4 vs 16 months, P<0.0001 for overall survival; 3.2 vs 7.9 months, P<0.0001 for progression-free survival). In a multivariate analysis, ctDNA status was independently associated with overall survival and progression-free survival (HR = 3.1, 95%CI = 1.9–5.0, P<0.0001; HR 2.6, 95%CI = 1.7–4.0, P<0.0001, respectively). Moreover, circulating tumor DNA significantly correlated with a higher number of liver metastases, the presence of lung and/or peritoneal metastases, tumor burden, and higher carbohydrate antigen 19–9 levels. This study supports the use of circulating tumor DNA as an independent prognostic marker for advanced pancreatic cancer.

Circulating tumour-derived KRAS mutations in pancreatic cancer cases are predominantly carried by very short fragments of cell-free DNA

BackgroundThe DNA released into the bloodstream by malignant tumours· called circulating tumour DNA (ctDNA), is often a small fraction of total cell-free DNA shed predominantly by hematopoietic cells and is therefore challenging to detect. Understanding the biological properties of ctDNA is key to the investigation of its clinical relevance as a non-invasive marker for cancer detection and monitoring. MethodsWe selected 40 plasma DNA samples of pancreatic cancer cases previously reported to carry a KRAS mutation at the ‘hotspot’ codon 12 and re-screened the cell-free DNA using a 4-size amplicons strategy (57 bp, 79 bp, 167 bp and 218 bp) combined with ultra-deep sequencing in order to investigate whether amplicon lengths could impact on the capacity of detection of ctDNA, which in turn could provide inference of ctDNA and non-malignant cell-free DNA size distribution. FindingsHigher KRAS amplicon size (167 bp and 218 bp) was associated with lower detectable cell-free DNA mutant allelic fractions (p < 0·0001), with up to 4·6-fold (95% CI: 2·6–8·1) difference on average when comparing the 218bp- and the 57bp-amplicons. The proportion of cases with detectable KRAS mutations was also hampered with increased amplicon lengths, with only half of the cases having detectable ctDNA using the 218 bp assay relative to those detected with amplicons less than 80 bp. InterpretationTumour-derived mutations are carried by shorter cell-free DNA fragments than fragments of wild-type allele. Targeting short amplicons increases the sensitivity of cell-free DNA assays for pancreatic cancer and should be taken into account for optimized assay design and for evaluating their clinical performance. FundingIARC; MH CZ – DRO; MH SK; exchange program between IARC and Sao Paulo medical Sciences; French Cancer League.

Abstract A60: Integrated proteogenomic characterization of pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with poor survival rates. Towards understanding the underlying molecular alterations that drive the PDAC oncogenesis, we conducted the first comprehensive proteogenomic analysis of 76 pancreatic tumors and 44 adjacent normal tissues (NATs). Proteomics, whole-genome sequencing, whole-exome sequencing, methylation, and RNA-seq were performed on these tissues to facilitate an integrated proteogenomic analysis and determine the impact of genomic alterations on protein expression. Genomic analysis showed that 89% of tumor samples had a detectable KRAS hotspot mutation, close to the mutation rate reported by TCGA. TP53 (56%), SMAD4 (18%), and CDKN2A (17%) rates were lower, but also close to those in the TCGA cohort, suggesting adequate tumor purity percentages in most of our samples. Additionally, we applied several deconvolution methods, including ESTIMATE and EDec, to estimate tumor purity and cell type composition using transcriptomics and DNA methylation data. These purity estimates correlated with KRAS variant allele frequency (VAF). RNA-based subtyping identified a balanced collection of subtypes consistent with previous reports in PDAC. However, gene expression and protein abundance were not correlated with the mean Spearman correlation of 0.23. Highly correlated pathways included pancreatic secretion, protein digestion and absorption, and allograft rejection, while pathways with low correlation encompassed oxidative phosphorylation and cytoskeleton organization during mitosis. Interestingly, mutations did not have a strong cis impact on the overall proteome level. However, we observed significant increases in BAX and DNMT1 protein levels associated with TP53 mutations. Tumor-normal analyses revealed tumor-specific gene and protein expression, including EMT, cell adhesion, and keratin genes. Proteomic analysis identified several druggable events in ERBB2, SYK, PAK1, PDPK1, and EPHA2. Using cell surface marker gene expression, we identified and classified our samples into four immune subtypes: NK- excluded, NK+ excluded, immune desert, and inflamed. PLEK, WAS, CD48, ALDOA, and ASPH had highly differential protein levels in inflamed tumors, among others. We further identified that protein levels of INTS14, SULF2, and PTPMT1, among others, were associated with patient survival, demonstrating the prognostic value of proteomics over RNA alone. Additionally, we are characterizing an additional 81 pancreatic tumors and 58 NATs using our deep proteogenomic analysis and post-translational modifications, aiming to verify the preliminary findings and to investigate the roles of cell signaling network and cell surface receptors on PDAC oncogenesis. This integrated proteogenomic characterization of PDAC will be a valuable resource for the community, paving the way for the discovery of novel therapeutic targets. Citation Format: Kyung Cho Cho, Ralph Hruban, Hui Zhang, Chen Huang, Bing Zhang, Daniel Cui Zhou, Li Ding, Emily Boja, Investigators from the Clinical Proteomic Tumor Analysis Consortium. Integrated proteogenomic characterization of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A60.

MET inhibitor resistance in patients with MET exon 14-altered lung cancers.

9006 Background: MET exon 14 alterations comprise a novel class of lung cancer drivers. MET tyrosine kinase inhibitors (TKIs) are active in patients with these cancers, but objective response rates (ORRs) are modest (~30%-40%). A subset of these cancers may harbor intrinsic resistance. Moreover, patients with initial benefit invariably develop acquired resistance. We set out to identify potential resistance mechanisms. Methods: We studied patients with stage IV MET exon 14-altered lung cancers who received a MET TKI. When feasible, tumor and/or plasma samples were collected, prioritizing paired pre- and post-TKI collection. Tumor samples underwent targeted mass spectrometry analysis (Nantomics) and DNA- (including MSK-IMPACT)/RNA-based (MSK-Fusion) next-generation sequencing (NGS). Plasma cfDNA underwent targeted NGS. ORR and progression-free survival (PFS) were assessed (RECIST v1.1). Results: 74 patients received a MET TKI (1 TKI n = 55; ≥2 TKIs n = 19). 91% received crizotinib as their 1st TKI. Pre-TKI MET levels in tumor tissue (range 0-2120 amol/µg) were associated with outcomes: ORR 63% (n = 7/11) and median PFS 6.9 mos with detectable MET vs ORR 0% (n = 0/5) and median PFS 4.6 mos with undetectable MET (HR for PFS 0.3). Pre-TKI RAS pathway activation was associated with response: ORR 0% (n = 0/6) with KRAS/NF1/RASA1 mutation vs ORR 29% (n = 25/87) in others. Similar outcomes were observed with pre-TKI KRAS expression (n = 16, all with detectable KRAS levels): ORR 0% (n = 0/2) in KRAS ≥700 amol/µg vs ORR 50% (n = 7/14) &lt; 700 amol/µg. Acquired resistance (Jackman criteria) was seen in 29 patients, 9 with paired pre-/post-treatment samples. On-target acquired resistance was found in 2/9 patients (22%): MET D1228N (n = 1), HGF amplification (n = 1). Potential off-target acquired resistance mechanisms were found in 5/9 pts (44%): KRAS G13V (n = 1), RASA1 S742* (n = 1), MDM2 amplification (n = 2), EGFR amplification (n = 1). Conclusions: Lack of MET expression or RAS pathway activation is associated with poor MET TKI outcomes in MET exon14-altered lung cancers. On-target acquired resistance is found in &lt; 25% of patients; HGF amplification is a novel mechanism. Off-target intrinsic/acquired resistance may be mediated by RAS/MDM2/EGFR pathway activation.

Analysis of Cell-Free DNA to Assess Risk of Tumoremia Following Endoscopic Ultrasound Fine-Needle Aspiration of Pancreatic Adenocarcinomas

Cellular and nuclear material from tumors disseminates into the bloodstream (tumoremia), but it is not clear whether medical procedures cause release of this material or contribute to formation of metastases. We performed a prospective study of blood samples from patients with pancreatic adenocarcinoma (PDAC) to determine whether endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) associates with markers of tumoremia. We obtained peripheral blood from 104 patients (35 with PDAC) before and after EUS-FNA of primary tumors; blood samples from 69 healthy individuals were used as controls. Plasma concentrations of cell-free DNA (cfDNA) were measured, and cfDNA and primary tumor samples were analyzed to detect activating mutations in KRAS. Potential development of tumoremia was defined by an increase in cfDNA of 2-fold or more, and/or detection of mutant KRAS in samples collected after FNA from patients whose blood samples did not contain detectable mutant KRAS before FNA. Peripheral blood concentrations of cfDNA were 1200 ng/ml (500-3300 ng/ml) before FNA vs 1400 ng/ml (900-4000 ng/ml) after FNA (P = .391). Tumoremia was detected in 10/35 patients (28.6%): 7 patients had a ≥2-fold increase in cfDNA concentration (20.6%) and 3 patients had circulating tumor DNA with KRAS mutations after FNA that were not detected before FNA (8.8%). New distant metastases were detected in 1.3 ± 0.82 patients with tumoremia vs 0.64±0.81 without (P = .0375). Overall mortality did not differ significantly between patients with tumoremia (10/10 deaths, 100%) vs those without (19/25 deaths, 76%) nor did survival times of deceased patients (13.3 months for patients with tumoremia; range, 5.8-14.9 months vs 11.1 months for patients without tumoremia; range, 5.5-14.5 months). However, 6 patients without tumoremia were alive at a mean 23.9 months after EUS-FNA (range, 19.9-25 months after EUS-FNA) vs none of the patients with tumoremia. In patients with PDAC, EUS-FNA associates with increased plasma concentration of cfDNA and increased detection of mutant KRAS after the procedure (markers of tumoremia and possible new distant metastasis). Although levels of cfDNA and activating mutations in KRAS are logical markers of tumoremia, they may not serve as the ideal biomarkers of this process. These findings are preliminary and do not indicate a need to modify current practice, yet further studies are needed.

Pancreatic cancer: Circulating Tumor Cells and Primary Tumors show Heterogeneous KRAS Mutations

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease. Circulating tumor cells (CTC) in the blood are hypothesized as the means of systemic tumor spread. Blood obtained from healthy donors and patients with PDAC was therefore subject to size-based CTC-isolation. We additionally compared Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations in pancreatic CTC and corresponding tumors, and evaluated their significance as prognostic markers. Samples from 68 individuals (58 PDAC patients, 10 healthy donors) were analyzed; CTCs were present in patients with UICC stage IA-IV tumors and none of the controls (p < 0.001). Patients with >3 CTC/ml had a trend for worse median overall survival (OS) than patients with 0.3–3 CTC/ml (P = 0.12). Surprisingly, CTCs harbored various KRAS mutations in codon 12 and 13. Patients with a KRASG12V mutation in their CTC (n = 14) had a trend to better median OS (24.5 months) compared to patients with other (10 months), or no detectable KRAS mutations (8 months; P = 0.04). KRAS mutations in CTC and corresponding tumor were discordant in 11 of 26 “tumor-CTC-pairs” (42%), while 15 (58%) had a matching mutation; survival was similar in both groups (P = 0.36). Genetic characterization, including mutations such as KRAS, may prove useful for prognosis and understanding of tumor biology.

Feasibility of multiplexed gene mutation detection in plasma samples of colorectal cancer patients by mass spectrometric genotyping.

Mutation analysis of circulating tumor DNA (ctDNA) has recently been introduced as a noninvasive tumor monitoring method. In this study, we tested the mass spectrometric-based MassARRAY platform for multiplexed gene mutation analysis of plasma samples from colorectal cancer (CRC) patients. A total of 160 patients, who underwent curative resection of either primary or metastatic CRC harboring KRAS mutations between 2005 and 2012, were included. Circulating DNA was isolated from plasma was analyzed on the MassARRAY platform with or without selective amplification of mutant DNA fragments. Tumor-specific KRAS mutations were detected in 39.6% (42/106) of patients with distant metastasis, and in 5.6% (3/54) of patients without distant metastasis. Selective amplification of the mutant allele increased sensitivity to 58.5% (62/106) for patients with distant metastasis, and 16.7% (9/54) for patients without distant metastasis. These mutation detection rates were no less than those of droplet digital polymerase chain reaction. Among patients with distant metastasis, detectable plasma KRAS mutations correlated with larger primary tumors and shorter overall survival rate (P = 0.014 and P = 0.003, respectively). In addition, activating PIK3CA mutations were detected together with KRAS mutations in two plasma samples. Taken together, massARRAY platform is a cost-effective, multigene mutation profiling technique for ctDNA with reasonable sensitivity and specificity.

High prevalence of mutantKRAS in circulating exosome-derived DNA from early-stage pancreatic cancer patients

Exosomes arise from viable cancer cells and may reflect a different biology than circulating cell-free DNA (cfDNA) shed from dying tissues. We compare exosome-derived DNA (exoDNA) to cfDNA in liquid biopsies of patients with pancreatic ductal adenocarcinoma (PDAC). Patient samples were obtained between 2003 and 2010, with clinically annotated follow up to 2015. Droplet digital PCR was performed on exoDNA and cfDNA for sensitive detection of KRAS mutants at codons 12/13. A cumulative series of 263 individuals were studied, including a discovery cohort of 142 individuals: 68 PDAC patients of all stages; 20 PDAC patients initially staged with localized disease, with blood drawn after resection for curative intent; and 54 age-matched healthy controls. A validation cohort of 121 individuals (39 cancer patients and 82 healthy controls) was studied to validate KRAS detection rates in early-stage PDAC patients. Primary outcome was circulating KRAS status as detected by droplet digital PCR. Secondary outcomes were disease-free and overall survival. KRAS mutations in exoDNA, were identified in 7.4%, 66.7%, 80%, and 85% of age-matched controls, localized, locally advanced, and metastatic PDAC patients, respectively. Comparatively, mutant KRAS cfDNA was detected in 14.8%, 45.5%, 30.8%, and 57.9% of these individuals. Higher exoKRAS MAFs were associated with decreased disease-free survival in patients with localized disease. In the validation cohort, mutant KRAS exoDNA was detected in 43.6% of early-stage PDAC patients and 20% of healthy controls. Exosomes are a distinct source of tumor DNA that may be complementary to other liquid biopsy DNA sources. A higher percentage of patients with localized PDAC exhibited detectable KRAS mutations in exoDNA than previously reported for cfDNA. A substantial minority of healthy samples demonstrated mutant KRAS in circulation, dictating careful consideration and application of liquid biopsy findings, which may limit its utility as a broad cancer-screening method.

Abstract 3960: Combined circulating tumour cell (CTC) and circulating tumor DNA (ctDNA) analysis of blood from patients with pancreatic cancer

Abstract Background The challenge to improve outcomes for patients diagnosed with advanced pancreatic cancer remains very real with only small improvements in median survival gained by the use of systemic chemotherapy and little improvement in 5-year survival over the past decades. The advent of next generation sequencing (NGS) of tumor nucleic acids has opened up the possibility of improving outcomes through personalized therapies selected on the basis of tumor genetics. Whilst NGS biomarkers can be measured in tumor biopsies sampled shortly before and after treatment this is often not practical for ethical, logistical and simple lack of availability. To circumvent problems associated with tumor sampling we have developed and evaluated blood-borne nucleic acids biomarkers for patients diagnosed with advanced pancreatic cancer. Approach We developed a NGS circulating free DNA (cfDNA) analysis pipeline based on the generation of whole genome NGS libraries followed by sequencing of over 600 cancer-associated genes using Agilent SureSelect. Using whole blood collected with Streck cell free DNA blood collection tubes (cfDNA BCT) we optimised combined circulating tumor cell (CTC) enrichment and cfDNA isolation. Quantitative measurements of KRAS mutations using both NGS and droplet digital PCR (ddPCR) were used to compare the tumor component present in both CTCs and cfDNA. We evaluated the overall approach using External Quality Assessment (EQA) controls and over 50 advanced pancreatic cancer patient samples. For CTC analysis we also compared epitope dependent (CellSearch) and independent (Parsortix) enrichment. Results After applying our NGS pipeline to 5 EQA genomic controls we identified all 14 known mutations correctly indicating high sensitivity and specificity. Both ddPCR and NGS identified KRAS mutations in patient cfDNA with a higher success rate seen for ddPCR consistent with the higher sensitivity of this methodology. From the NGS output additional mutations were detected in samples which either harboured or lacked detectable KRAS mutations. Consistent with previous observations CellSearch CTCs were detected at low levels in around 20% of patients with a similar frequency seen in initial analysis of CTCs obtained by epitope independent enrichment (Parsortix). In general KRAS mutations were detected at a higher level in patient cfDNA compared to enriched CTCs although some patients showed detectable CTC KRAS mutations with no KRAS mutations detected in their cfDNA by either ddPCR or NGS. Ongoing analysis is aimed at establishing if the molecular observations correlate with clinical outcome. Conclusion Combined CTC enrichment and cfDNA isolation is readily achievable using a single Streck cfDNA BCT. Results indicate that combined CTC and cfDNA analysis is more sensitive than either approach alone. Citation Format: Ged Brady, Dominic Rothwell, Mahmood Ayub, Nigel Smith, Sumitra Mohan, Jakub Chudziak, Kyaw Aung, Richard Hubner, Crispin Miller, Alison Backen, Hui Sun Leong, Sakshi Gulati, Chang Sik Kim, Angela Lamarca, Mairéad McNamara, Juan Valle, Caroline Dive. Combined circulating tumour cell (CTC) and circulating tumor DNA (ctDNA) analysis of blood from patients with pancreatic cancer. [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 3960.

Abstract 3137: NGS-based detection of KRAS hotspot mutations in plasma cell-free DNA of pancreatic cancer cases

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by hotspot mutations in the KRAS gene (codons 12, 13 or 61) in 85-90% of cases. Codon 12 KRAS mutations have been detected in pancreatic juice, blood and stool samples from pancreatic cancer cases and represent promising biomarkers for early detection. However, the proportion of tumor-derived KRAS mutations in cell-free DNA fragments (cfDNA) has shown large variations, probably because of the heterogeneity in biosamples and assays tested. Deep sequencing technologies (NGS) allow the identification of low-abundance somatic variants, but have not previously been applied to the detection of KRAS hotspot mutations in cfDNA of PDAC cases. Moreover, variant calling methods have rarely been tested against cancer-free individuals so the proportion of false positives is unknown. We investigated whether deep sequencing of KRAS mutations at codons 12, 13 and 61 in plasma samples could represent a robust assay to distinguish pancreatic cancer from chronic pancreatitis and healthy controls. Methods: We developed an Ion Torrent-based NGS KRAS assay (partial exons 2 and 3, totalling 208bp) to screen cfDNA from plasma samples of 461 PDAC cases, 154 individuals with chronic pancreatitis and 421 healthy controls. cfDNA extraction (&amp;gt;4ng) and sequencing (&amp;gt;1000x coverage on average, and absence of systematic high sequencing error rates on the 208bp) performed well on 431 (93%) PDAC cases, 138 (90%) chronic pancreatitis, and 388 (95%) controls. We fit a robust negative-binomial regression to estimate the distribution of the sequencing errors at each DNA bp position and identified outlying samples, which were considered as KRAS positive when q-value&amp;lt;10-3. We also estimated the detection threshold of our assay using serial dilutions of DNA from SW480 KRAS mutated cell-line (p.G12V) in wild-type DNA. Results: Sequencing of the serial dilutions of KRAS p.G12V mutated DNA indicated a detection threshold at a minor allele frequency of 0.2%. KRAS mutations in cfDNA were detected in 83 (19.3%) PDAC cases (73 on codon 12; 8 on codon 61; 1 on codon 13; and 1 multiple codons, i.e., similar in proportions as reported in tumor tissue from the International Cancer Genomic Consortium); 3 (2.2%) chronic pancreatitis (all on codon 12); and 8 (2.1%) healthy controls (4 on codon 12 and 4 on codon 61). Stage was significantly associated with the proportion of detected mutations in cancer cases (chi-squared p = 0.0005): the proportions of cases with detectable KRAS mutations in plasma were 7.9%, 14.9%, and 31.1% for local, regional, and advanced stages, respectively. Conclusions: The NGS-based KRAS mutation screening is a sensitive approach to detect low allelic fraction in plasma cfDNA, although its utility for early detection is still limited. However, it has the capacity to identify specific KRAS mutations, which could be useful in a panel of other non-invasive biomarkers. Citation Format: Florence Le Calvez-Kelm, Matthieu Foll, Magdalena B. Wozniak, Geoffroy Durand, Priscilia Chopard, Maroulio Pertesi, Tiffany Delhomme, Ivana Holcatova, Lenka Foretova, Vladimir Janout, Eleonora Fabianova, Maxime P. Vallée, Paul Brennan, James D. McKay, Graham Byrnes, Ghislaine Scélo. NGS-based detection of KRAS hotspot mutations in plasma cell-free DNA of pancreatic cancer cases. [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 3137.

Clinical utility of KRAS status in circulating plasma DNA compared to archival tumour tissue from patients with metastatic colorectal cancer treated with anti-epidermal growth factor receptor therapy

BackgroundCirculating cell-free DNA (cfDNA) in plasma is a mixture of DNA from malignant and normal cells, and can be used as a liquid biopsy to detect and quantify tumour specific mutations e.g. KRAS. We investigated the clinical value of KRAS mutations when detected in plasma compared to tumour in patients from metastatic colorectal cancer (mCRC) prior to anti-epidermal growth factor receptor (anti-EGFR) therapy. Secondly, we investigated the concentration of total cfDNA in relation to clinical outcome. Patients and methodsPatients were resistant to 5-FU, oxaliplatin and irinotecan and treated with 3rd line irinotecan (180mg/m2) and cetuximab (500mg/m2) q2w in a prospective phase II trial. The study was conducted prior to implementation of KRAS as selection criteria. Plasma was obtained from a pre-treatment EDTA blood-sample, and the total cfDNA, and KRAS mutations were quantified by an in-house qPCR method. Results are presented according to REMARK. ResultsOne-hundred-and-forty patients were included. Thirty-four percent had detectable KRAS mutations in the tumour, compared to 23% in plasma. KRAS detection in archival tumour tissue showed no correlation to survival, whereas plasma KRAS status remained a strong predictive and prognostic factor in multivariate analysis (Hazard Ratio (HR)=2.98 (95% CI 1.53–5.80, p=0.001) and 2.84 (1.46–5.53, p=0.002), for OS and PFS, respectively). Combining the information of total cell free DNA levels and plasma KRAS mutation status, produced an additional prognostic effect. ConclusionThe value of clinically relevant mutations could be improved by performing the analysis on circulation plasma DNA rather than archival tumour tissue.

Abstract 5239: KRAS wild-type status as detected by circulating tumor DNA analysis may be a prognostic or predictive factor for clinical benefit in patients with unresectable, locally advanced or metastatic pancreatic cancer (PC) treated with the MEK inhibitor refametin

Abstract Background: Mutationally-activated KRAS is present in 90% of pancreatic ductal adenocarcinoma (PDAC) and may represent an early genetic driver, being commonly found in low-grade pancreatic lesions (Eser et al. 2014 BJC 111:817). Refametinib is a potent oral allosteric MEK 1/2 inhibitor with both single-agent activity and synergistic activity in combination with gemcitabine in preclinical models of pancreatic cancer (PC). A Phase 1B/2 study in patients with locally advanced, unresectable or metastatic PC and no prior systemic therapy was conducted and recently reported (Van Laethem et al., ASCO 2014; NCT01251640). We report here on the exploratory biomarker findings from this study. Methods: KRAS mutational analysis was conducted via liquid biopsy at baseline on circulating tumor DNA (ctDNA) by BEAMing (Sysmex-Inostics) as well as circulating micro RNA (miRNA) from plasma collected at baseline and post-dose and analyzed by qPCR using an Exiqon panel of 752 miRNAs and an innovative data preprocessing method for normalization and imputation of undetermined values. Tumor molecular characterization was performed on archival tumor tissue and included targeted tumor gene next-generation sequencing with FOUNDATION ONE and the analysis of Ki67 proliferation index. Results: Samples for biomarker analysis were obtained from 69 treated patients. Forty-six (67%) had detectable KRAS mutations by liquid biopsy. KRAS G12D, G12V and G12R were the most frequent mutations. Interestingly, KRAS wild-type patients had better efficacy outcomes compared to mutant KRAS patients (mut/WT, respectively): overall response rate 15%/30% (OR 2.4, p = 0.147), median progression-free survival (mPFS) 3.7/8.8 mo (HR 0.32, p = 0.001), and overall survival (OS) 7.1/18.2 mo (HR 0.28, p = 0.001). There was a trend correlating KRAS mutant allele frequency with response. The CA19.9 levels correlated with KRAS mutational status. Tumor exome sequencing was performed from 16 patients, 15 of which had a KRAS mutation (G12D or G12V). The discordancy rate compared to BEAMing KRAS data was 26% (4/15). Conclusions: The high prevalence of KRAS mutations in patients with PC has been confirmed using BEAMing technology. In this study, there was an association between improved mPFS and OS in KRAS WT patients. Together with lower baseline levels of CA19.9 in the KRAS WT cohort, we conclude that liquid biopsy may be an approach to identify prognostic or predictive markers in PDAC treated with refametinib and gemcitabine. This hypothesis is sustained by the finding that poor clinical response showed increasing allele frequency of mutant KRAS. These results require confirmation in a larger trial. Citation Format: Michael Teufel, Jean-Luc Van Laethem, Hanno Riess, Marius Giurescu, Vittorio L. Garosi, Anke Schulz, Richardus Vonk, Henrik Seidel, Joachim Reischl, Barrett H. Childs. KRAS wild-type status as detected by circulating tumor DNA analysis may be a prognostic or predictive factor for clinical benefit in patients with unresectable, locally advanced or metastatic pancreatic cancer (PC) treated with the MEK inhibitor refametin [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 5239. doi:10.1158/1538-7445.AM2015-5239