KRAS ctDNA Research Articles

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.

Preoperative detection of KRAS mutated circulating tumor DNA is an independent risk factor for recurrence in colorectal cancer

Preoperative ctDNA status in relation to recurrence in cases of CRC remains unclear. We examined preoperative ctDNA detection by targeting KRAS gene mutations as a predictive marker for recurrence after CRC surgery. We measured the preoperative KRAS mutated ctDNA status and analyzed the correlation with clinicopathologic features of 180 patients that underwent surgery for CRC. We studied the association between preoperative KRAS mutated ctDNA and postoperative recurrence in patients (n = 150) that underwent radical surgery. KRAS mutated ctDNA was detected in 59 patients (32.8%). Median mutant allele frequency of KRAS in ctDNA was 0.20%. KRAS status in ctDNA and lymph node metastasis and distant metastasis were not significantly different. Among patients that underwent radical resection, recurrence occurred in 21 (14.0%, median follow-up 24 months). In Kaplan–Meier analysis, preoperative detection of KRAS mutated ctDNA was associated with inferior recurrence-free interval (RFI) (p = 0.002) and recurrence-free survival (RFS) (p = 0.025). In a multivariate Cox proportional hazards model, preoperative detection of KRAS mutated ctDNA was an independent factor related to both RFI (HR = 3.08; p = 0.012) and RFS (HR = 2.18; p = 0.044). Preoperative measurement of KRAS mutated ctDNA could be useful to decide postoperative treatment.

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.

Abstract A07: Sequential ctDNA analysis detected preclinical relapse in patients with metastatic colorectal cancer from the Exactis trial (NCT00984048)

Abstract The Exactis trial (NCT00984048), assessed plasma circulating-tumor DNA (ctDNA) mutations and whether they correlated with their relative levels in metastatic tumor biopsies in 50 mCRC enrolled patients undergoing first-line treatment. Exactis is a Canadian National Centre of Excellence in Personalized Medicine and clinical trials. Two-hundred and twenty-two plasma ctDNA samples collected at baseline, on treatment, and at time of clinical resistance (median 4 samples per patient) were sequenced and analyzed using the Contextual Genomics FOLLOW ITTM assay with QUALITY NEXUSTM bioinformatics analytical pipeline. This panel assesses hotspot mutations and frequently mutated regions in 30 commonly mutated cancer genes. Patient overall objective response was based on RECIST v1.0 criteria. We detected ctDNA mutations in at least one plasma timepoint in 92% (46/50) of patients and in multiple timepoints in 76% (38/50), including mutations in KRAS or NRAS (52%), PIK3CA (26%), BRAF (6%), and TP53 (74%). In 3 of the 4 patients with no detectable mutations, exome sequencing of the tumor revealed no mutations covered by the ctDNA assay. The other had both a KRAS and PIK3CA mutation in the tumor data; both were present in the FOLLOW ITTM sequencing data but were below the clinically validated threshold for the assay. Of interest, 8 out of 26 patients with ctDNA KRAS mutations detected (31%) had a G12D or G12C variant, which may have implications for new targeted therapeutics. We also observed a trend toward enrichment of KRAS/NRAS mutations in nonresponder (NR) compared to responder (R) patients (16/23 NR versus 10/23 R, p=0.07, Chi-square test). Furthermore, the occurrence of PIK3CA alone or in combination with KRAS mutations was significantly higher in the NR patients (10/23 NR versus 3/23 R p=0.02; and 8/23 NR versus 1/23 R p=0.0092, Chi-square test). Within the NR patient population, we found that 17 out of 23 (74%) patients harbored detectable ctDNA mutations preceding progression detection based on CT scan imaging, highlighting the potential of liquid biopsies to monitor disease progression in mCRC. Our study showed that the FOLLOW ITTM assay is capable of detecting mutations in CRC driver genes using liquid biopsy within a clinical trial setting. The identification of plasma ctDNA mutations in this context could allow for re-evaluation and potential change in management before clinical or CT scan detected relapse has occurred. In addition, the detection of ctDNA PIK3CA and KRAS mutations enrichment in the NR population warrants further validation and investigation for its clinical utility for patient stratification. Citation Format: Melissa K. McConechy, Suzan McNamara, Mathilde Couetoux du Tertre, Karen Gambaro, Maud Marques, Salem Malikic, Ka Mun Nip, Sonal Brahmbhatt, Adrian Kense, Kevin Tam, Rosalia Aguirre Hernandez, Ruth Miller, Madeline Couse, Jas Khattra, David G. Huntsman, Gerald Batist. Sequential ctDNA analysis detected preclinical relapse in patients with metastatic colorectal cancer from the Exactis trial (NCT00984048) [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A07.

Performance of four platforms for KRAS mutation detection in plasma cell-free DNA: ddPCR, Idylla, COBAS z480 and BEAMing

Multiple platforms are commercially available for the detection of circulating cell-free tumour DNA (ctDNA) from liquid biopsies. Since platforms have different input and output variables, deciding what platform to use for a given clinical or research question can be daunting. This study aimed to provide insight in platform selection criteria by comparing four commercial platforms that detect KRAS ctDNA hotspot mutations: Bio-Rad droplet digital PCR (ddPCR), BioCartis Idylla, Roche COBAS z480 and Sysmex BEAMing. Platform sensitivities were determined using plasma samples from metastatic colorectal cancer (mCRC) patients and synthetic reference samples, thereby eliminating variability in amount of plasma analysed and ctDNA isolation methods. The prevalence of KRAS nucleotide alterations was set against platform-specific breadth of target. Platform comparisons revealed that ddPCR and BEAMing detect more KRAS mutations amongst mCRC patients than Idylla and COBAS z480. Maximum sample throughput was highest for ddPCR and COBAS z480. Total annual costs were highest for BEAMing and lowest for Idylla and ddPCR. In conclusion, when selecting a platform for detection of ctDNA hotspot mutations the desired test sensitivity, breadth of target, maximum sample throughput, and total annual costs are critical factors that should be taken into consideration. Based on the results of this study, laboratories will be able to select the optimal platform for their needs.

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.

KRAS Mutant Allele Fraction in Circulating Cell-Free DNA Correlates With Clinical Stage in Pancreatic Cancer Patients.

Background: The research on circulating tumor DNA (ctDNA) in pancreatic cancer (PC) has emerged recently. Although the detection rate of the KRAS mutation in ctDNA was relatively consistent with that in tumor tissue, whether the KRAS mutant allele fraction (MAF) differed was still not reported. So far, the clinical application of ctDNA detection in PC remains inconclusive.Methods: Plasma samples were collected from 110 PC and 52 pancreatic benign (PB) disease patients. The detection of KRAS mutation in ctDNA was performed using droplet digital PCR and compared with that in matched tumor tissue. We assessed the utility of KRAS MAFs in ctDNA and tissue for pancreatic malignancy assessment.Results: We found that KRAS MAF in ctDNA of PC patients was higher than that of PB patients, and was obviously associated with tumor staging and distant metastasis. However, KRAS MAF in ctDNA was significantly different from that in matched tissue. KRAS MAF in tumor tissue had no significant correlation with the clinical status. In addition, a ROC curve analysis revealed that mutant KRAS ctDNA combined with CA19-9 could increase the sensitivity rate of early-stage PC prediction, compared with CA19-9 test alone.Conclusion: The MAF of KRAS in ctDNA was related to the clinical stage of PC (p = 0.001). Mutant KRAS ctDNA could improve the sensitivity in early diagnosis of PC as a complement to CA19-9. Our study suggested that KRAS mutation in ctDNA could be a valuable circulating biomarker for the malignancy assessment in PC.

Circulating tumour DNA (ctDNA) as a biomarker in metachronous melanoma and colorectal cancer- a case report

BackgroundCirculating tumour DNA (ctDNA) has emerged as a promising blood-based biomarker for monitoring disease status of patients with advanced cancers. The presence of ctDNA in the blood is a result of biological processes, namely tumour cell apoptosis and/or necrosis, and can be used to monitor different cancers by targeting cancer-specific mutation.Case presentationWe present the case of a 67 year old Caucasian male that was initially treated with BRAF inhibitors followed by anti-CTLA4 and then anti-PD1 immunotherapy for metastatic melanoma but later developed colorectal cancer. The kinetics of ctDNA derived from each cancer type were monitored targeting BRAF V600R (melanoma) and KRAS G13D (colon cancer), specifically reflected the status of the patient’s tumours. In fact, the discordant pattern of BRAF and KRAS ctDNA was significantly correlated with the clinical response of melanoma to pembrolizumab treatment and progression of colorectal cancer noted by PET and/or CT scan. Based on these results, ctDNA can be used to specifically clarify disease status of patients with metachronous cancers.ConclusionsUsing cancer-specific mutational targets, we report here for the first time the efficacy of ctDNA to accurately provide a comprehensive outlook of the tumour status of two different cancers within one patient. Thus, ctDNA analysis has a potential clinical utility to delineate clinical information in patients with multiple cancer types.

Highlights of This Issue

The FGFR pathway is thought to drive oncogenic progression of many tumor types. Bahleda and colleagues report on the results of a first-in-human phase I trial of erdafitinib (JNJ-42756493), a potent, oral pan-FGFR tyrosine kinase inhibitor, conducted on patients with advanced-stage disease with known genetic activation of the FGFR pathway. This agent was well-tolerated and showed promising efficacy, especially in urothelial carcinoma and cholangiocarcinoma patients with FGFR alterations. These results have the potential to influence future treatment of patients with FGFR positive tumors.Myelofibrosis is morphologically characterized by the presence of atypical megakaryocytes and bone marrow fibrosis. To determine whether elimination of these abnormal megakaryocytes has therapeutic benefit for myelofibrosis patients, Gangat and colleagues conducted a phase 1 study of alisertib, an aurora kinase A inhibitor and megakaryocyte differentiation agent. Alisertib was well-tolerated and provided a modest clinical benefit for spleen size and symptom burden. Moreover, alisertib normalized megakaryocyte morphology and reduced fibrosis in a substantial number of patients. This study underscores the role of megakaryocytes in myelofibrosis and highlights the feasibility of targeting this lineage in this disease.Cancer vaccines have long been considered a promising tool for cancer therapy. However, many potential vaccines have been ineffective in clinical trials, in part due to the immunosuppressive cancer microenvironment. In a phase I trial, Gatti-Mays and colleagues examined BN-CV301, a modified cancer vaccine expressing MUC1 and CEA tumor antigens, as well as costimulatory molecules. Treatment-related adverse events were relatively minor. Patients with mutant KRAS responded particularly well to this regimen – five out of six patients showed either a partial response or stable disease after BN-CV301 vaccination. This improved vaccine shows promise for further clinical evaluation.Pancreatic ductal adenocarcinoma is a highly lethal cancer due, in part, to a high rate of recurrence. Groot and colleagues developed and validated an assay to detect KRAS hotspot mutations in circulating tumor DNA (ctDNA) in a CLIA laboratory setting. Detection of mutant KRAS in ctDNA at the time of surgery was associated with a greater risk of recurrence, while rising mutant KRAS levels in ctDNA provided an early prediction of recurrence. In several instances, the detection of mutant KRAS in ctDNA outperformed the measurement of CA 19-9 in serum. This liquid biopsy test for mutant KRAS shows great promise for monitoring disease in pancreatic cancer patients, potentially leading to earlier intervention.

Circulating Tumor DNA as a Clinical Test in Resected Pancreatic Cancer.

In research settings, circulating tumor DNA (ctDNA) shows promise as a tumor-specific biomarker for pancreatic ductal adenocarcinoma (PDAC). This study aims to perform analytical and clinical validation of a KRAS ctDNA assay in a Clinical Laboratory Improvement Amendments (CLIA) and College of American Pathology-certified clinical laboratory. Digital-droplet PCR was used to detect the major PDAC-associated somatic KRAS mutations (G12D, G12V, G12R, and Q61H) in liquid biopsies. For clinical validation, 290 preoperative and longitudinal postoperative plasma samples were collected from 59 patients with PDAC. The utility of ctDNA status to predict PDAC recurrence during follow-up was assessed. ctDNA was detected preoperatively in 29 (49%) patients and was an independent predictor of decreased recurrence-free survival (RFS) and overall survival (OS). Patients who had neoadjuvant chemotherapy were less likely to have preoperative ctDNA than were chemo-naïve patients (21% vs. 69%; P < 0.001). ctDNA levels dropped significantly after tumor resection. Persistence of ctDNA in the immediate postoperative period was associated with a high rate of recurrence and poor median RFS (5 months). ctDNA detected during follow-up predicted clinical recurrence [sensitivity 90% (95% confidence interval (CI), 74%-98%), specificity 88% (95% CI, 62%-98%)] with a median lead time of 84 days (interquartile range, 25-146). Detection of ctDNA during postpancreatectomy follow-up was associated with a median OS of 17 months, while median OS was not yet reached at 30 months for patients without ctDNA (P = 0.011). Measurement of KRAS ctDNA in a CLIA laboratory setting can be used to predict recurrence and survival in patients with PDAC.

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

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

Sequential assessments of KRAS-mutated circulating tumor DNA in longitudinal monitoring enable the prediction of prognosis and therapeutic responses in patients with pancreatic cancer.

e15712 Background: Liquid biopsy enables the detection of circulating tumor DNA (ctDNA) levels, including KRAS-mutated ctDNA, which is considered a predictive biomarker for pancreatic cancer. This study aimed to evaluate the significance of sequential KRAS ctDNA assessments in longitudinal monitoring. Methods: A total of 422 blood samples were collected from 78 patients undergoing treatments for localized and metastatic pancreatic ductal adenocarcinoma. KRAS ctDNA levels was determined by droplet digital polymerase chain reaction. Longitudinal monitoring of KRAS ctDNA was performed to assess its significance for predicting recurrence and prognosis and evaluating therapeutic responses to chemotherapy. Results: In 39 patients who underwent surgery for potentially resectable tumors, sequential assessments of KRAS ctDNA in longitudinal monitoring was significantly associated with prognosis ( P &lt; 0.001). In 39 patients who did not undergo surgery, sequential assessments of KRAS ctDNA was a predictive factor for prognosis ( P = 0.005). Multivariate analysis revealed that detection of KRAS ctDNA in longitudinal monitoring was the only independent prognostic factor regardless of tumor resection ( P&lt; 0.001). Longitudinal monitoring revealed the significance of sequential assessments of KRAS ctDNA within a short period. The presence of KRAS ctDNA in sequential assessments within 1 year after surgery showed significant association with prognosis irrespective of recurrence ( P&lt; 0.001). The presence of KRAS ctDNA in sequential assessments within 6 months was significantly correlated with therapeutic responses in the first line chemotherapy ( P&lt; 0.001). Conclusions: Our study showed for the first time that sequential assessments of KRAS ctDNA levels within a short period enabled the prediction of prognosis and therapeutic response in patients with pancreatic cancer.

Circulating tumor DNA-based mutational landscape of advanced colorectal cancer.

e15034 Background: Alterations in oncogenic driver genes are extensively tested to guide anticancer treatment in colorectal cancer (CRC). As the repertoire of therapeutic targets continue to grow, we are interested to interrogate the possibility of resorting to circulating tumor DNA (ctDNA) where tissue samples are not readily available. Methods: Targeted next generation sequencing (NGS) was performed on ctDNA obtained from patients with advanced CRC using a 150-gene panel. Mutational profiles of ctDNA were compared with those detected in tissue samples from the 3D Medicine database as well as The Cancer Genome Atlas (TCGA) database. Genomic aberrations such as single nucleotide variations (SNVs), insertions/deletions, copy number variations, gene rearrangement and fusions were analyzed. Results: Notably, 244 of 256 samples (95.3%) were ctDNA-positive, as indicated by a maximum somatic allele frequency (MSAF) of &gt; 0%. At least one reportable genomic alteration was detected in 96.5% of all cases, with an average of 8.12 mutations per case. The most frequently mutated genes were TP53 (47%), APC (42%), KRAS (32%) in ctDNA. A similar trend was also observed for SNVs, where TP53 (46.9% vs. 77.6% vs. 53.8%), APC (41.8% vs. 66.0% vs. 73.0%), and KRAS (30.5% vs. 48.4% vs. 43.5%) had the highest mutation frequencies across three data sets (ctDNA, tissues and the TCGA database). The SNV profiles of therapeutically relevant genes for CRC were also largely consistent between ctDNA and the tissue samples included in the 3D Medicine database: BRAF (7.0% vs. 8.7%), KRAS (30.5% vs. 48.4%), NRAS (2.7 vs. 3.1%), ERBB2 (5.1% vs. 5.2%), MLH1 (0.8% vs. 2.9%), MSH2 (2.7% vs. 3.1%), and MSH6 (3.1% vs. 3.7%). Patients harboring copy number gains had a significantly higher level of MSAF than those free of gene amplifications (p &lt; 0.0001). Conclusions: Mutational landscape of ctDNA in general correlated with that detected in prior tissue samples as well as the TCGA database, supporting a role for ctDNA testing as a complementary approach to tissue testing in CRC.

Su1371 PROSPECTIVE EVALUATION OF CLINICAL, LABORATORY, AND TUMOR-RELATED CHARACTERISTICS ASSOCIATED WITH HEMATOGENOUS TUMOR SEEDING FOLLOWING EUS FNA OF PANCREATIC DUCTAL ADENOCARCINOMA

Occult dissemination of tumor cellular and nuclear material into the bloodstream is well-recognized and has been reported for a variety of tumor types, including pancreatic ductal adenocarcinoma (PDAC). These cells and nuclear material can be detected in the blood as circulating tumor cells (CTCs) or cell-free DNA (cfDNA) as a means of “liquid biopsy” and their presence is referred to as “tumoremia.” Limited data suggest that various biopsy procedures and surgery can also precipitate tumoremia. There are no published data indicating the risk of hematogenous tumor seeding following EUS FNA and no data clarifying whether any factors may be associated with its occurrence. In PDAC patients, we aimed to prospectively evaluate peripheral blood to determine the risk of EUS FNA-induced tumoremia and to identify the associated clinical, laboratory, and tumor demographic features. Peripheral blood from 35 patients with PDAC was collected before and following primary tumor EUS FNA. Plasma cfDNA concentrations and KRAS 12/13 mutation status were evaluated. Tumor seeding was defined by a ≥2-fold increase in cfDNA and/or conversion from circulating tumor deoxyribonucleic acid (ctDNA) KRAS mutant non-detectable to detectable. Factors that were evaluated to determine their correlation with tumoremia included age, gender, BMI, ECOG status, serum glucose level, CA 19-9 concentration, tumor location, tumor diameter, tumor stage, resectability status, number of FNA passes, infiltration of major arteries and veins, presence of IPMN, mortality, survival duration, pre-FNA cfDNA level, and the tumor allelic ratio. Based on our pre-defined metrics, either a ≥2-fold increase in the total cfDNA concentration (n=7; 20.6%), and/or conversion in ctDNA mutant detection status (n=3; 8.8%) primary tumor EUS FNA-induced hematogenous tumor seeding occurred in 10/35 (28.6%) patients. A broad spectrum of clinical, laboratory, and tumor demographic features were evaluated, none of which statistically correlated with a ≥2-fold increase in cfDNA, other than the number of major arteries and/or veins infiltrated by the primary tumor. (Table) The only feature that correlated with the conversion in KRAS mutation status was the CA 19-9 level. For the combined group (n=10), who met either metric for FNA-induced tumoremia, there were no characteristics that correlated with these endpoints. Our data indicate that the occurrence of hematogenous tumor seeding associated with EUS FNA at the present time, cannot be predicted based upon an evaluation of an extensive array of clinical, laboratory, and tumor related characteristics. Additional studies are needed to determine the clinical impact of EUS FNA-induced tumor seeding as well as to verify whether any predictive features can be identified to help predict its occurrence.Tabled 1≥2 fold increase in cfDNA concentrations &/or ctDNA- WT to ctDNA-MT Status (n=10)Remaining Cohort (n=25)P ValueAge (years)70.6 ± 6.269.4 ± 9.20.699Gender (female)4 (40.0%)14 (56.0%)0.471BMI24.8 ± 5.227.8 ± 6.90.229ECOG (≥1)1 (10.0%)9 (36.0%)0.407Glucose level (median; mg/dL)105.0 (98.0-107.0)121.5 (104.5-146.5)0.089Ca19-9 (median; U/mL)374.0 (222.0-1701.0)581.0 (74.5-1617.5)0.969Ca19-9 (>55 U/mL)9 (90.0%)19 (76.0%)0.645Tumor location (head)8 (80.0%)20 (80.0%)0.9Tumor diameter (long axis; mm)32.5 ± 6.531.9 ± 9.70.863Tumor diameter (short axis; mm)28.4 ± 7.026.4 ± 7.80.481Tumor stage (III/IV)5 (50.0%)11 (44.0%)0.9Tumor stage (I/II/III)8 (80.0%)18 (72.0%)0.9Tumor (resectable/borderline)5 (50.0%)14 (56.0%)0.9EUS FNA passes (median)5.5 (4.0-8.0)5.0 (4.8-6.0)0.739Infiltrate major artery (yes)4 (40.0%)11 (44.0%)0.9Infiltrate major vein (yes)4 (40.0%)15 (60.0%)0.454Infiltrate major artery or vein (yes)6 (60.0%)16 (64.0%)0.9# of major arteries/veins involved1.0 (0.0-2.0)2.0 (0.0-3.0)0.321Adenocarcinoma (non-IPMN)7 (70.0%)18 (72.0%)0.9Mortality (yes)6 (60.0%)14 (56.0%)0.9Time to mortality (median; months)6.4 (4-12.3)7.2 (4.4-11.3)0.9Perip Pre-FNA cfDNA level (ng/ml)0.6 (0.2-2.8)1.3 (0.6-3.5)0.201Allelic ratio (median; primary tumor)10.4 (1.5-23.4)18.9 (5.3-28.9)0.317 Open table in a new tab

Plasma KRAS as a biomarker for pancreatic ductal adenocarcinoma (PDAC).

316 Background: PDAC needs validated diagnostic biomarkers for early detection and predictive markers for outcome. As 95% of PDACs harbor KRAS mutations (mKRAS), circulating tumor DNA (ctDNA) has potential utility in PDAC. We assessed the ability to detect and correlate mKRAS in a metastatic PDAC cohort from Memorial Sloan Kettering Cancer Center. Methods: 10 mL of whole blood was collected. cfDNA was extracted with QIAmp or QIAsymphony DNA extraction kits (Qiagen, Valencia, CA). Directed (KRAS G12D, G12R, G12V, Q61H) or multiplex (G12A, G12C, G12D, G12R, G12S, G12V, G13D) digital droplet PCR (ddPCR) was performed with Raindrop Plus (Raindance Technologies, Billerica, MA) or QX200 (BioRad, Hercules, CA) ddPCR systems. Number and size of liver, lung and lymph node metastases, peritoneal disease (mild, moderate, severe), ascites (trace, small, large) and bone mets (Y/N) were assessed by CT scan. Results: See table. 21 (55%) had detectable ctDNA (ctDNA(+)) with mean mutant allele fraction of 4.5% (0.015-36.8). ctDNA (+) vs (-) PFS and OS from collection were 6.9 and 8.4 months vs. 9.9 and 10.5 (p=0.89 for both). CA19-9, PFS and OS did not correlate with ctDNA tertile (p=0.15, 0.54 &amp; 0.50). On treatment and disease activity were not associated with ctDNA status (p= 0.20 &amp; 0.60). Number and size of liver mets were associated with ctDNA (+) (p=0.006 &amp; 0.007). Conclusions: ctDNA KRAS detection was measurable in metastatic disease with rates consistent with other PDAC reports. Median PFS, OS were lower in ctDNA (+) group but not statistically significant in this diverse cohort. Number and size of liver metastases were significantly higher in ctDNA (+). Future study should focus on practice changing applications with standardized collection, intra-patient comparisons and role of liver disease burden. We have initiated studies to evaluate plasma KRAS prior to and during treatment to address its value as a predictive assay and explore factors affecting ctDNA detection. [Table: see text]

Mutant KRAS Circulating Tumor DNA Is an Accurate Tool for Pancreatic Cancer Monitoring.

Many new pancreatic cancer treatment combinations have been discovered in recent years, yet the prognosis of pancreatic ductal adenocarcinoma (PDAC) remains grim. The advent of new treatments highlights the need for better monitoring tools for treatment response, to allow a timely switch between different therapeutic regimens. Circulating tumor DNA (ctDNA) is a tool for cancer detection and characterization with growing clinical use. However, currently, ctDNA is not used for monitoring treatment response. The high prevalence of KRAS hotspot mutations in PDAC suggests that mutant KRAS can be an efficient ctDNA marker for PDAC monitoring. Seventeen metastatic PDAC patients were recruited and serial plasma samples were collected. CtDNA was extracted from the plasma, and KRAS mutation analysis was performed using next-generation sequencing and correlated with serum CA19-9 levels, imaging, and survival. Plasma KRAS mutations were detected in 5/17 (29.4%) patients. KRAS ctDNA detection was associated with shorter survival (8 vs. 37.5 months). Our results show that, in ctDNA positive patients, ctDNA is at least comparable to CA19-9 as a marker for monitoring treatment response. Furthermore, the rate of ctDNA change was inversely correlated with survival. Our results confirm that mutant KRAS ctDNA detection in metastatic PDAC patients is a poor prognostic marker. Additionally, we were able to show that mutant KRAS ctDNA analysis can be used to monitor treatment response in PDAC patients and that ctDNA dynamics is associated with survival. We suggest that ctDNA analysis in metastatic PDAC patients is a readily available tool for disease monitoring. Avoiding futile chemotherapy in metastatic pancreatic ductal adenocarcinoma (PDAC) patients by monitoring response to treatment is of utmost importance. A novel biomarker for monitoring treatment response in PDAC, using mutant KRAS circulating tumor DNA (ctDNA), is proposed. Results, although limited by small sample numbers, suggest that ctDNA can be an effective marker for disease monitoring and that ctDNA level over time is a better predictor of survival than the dynamics of the commonly used biomarker CA19-9. Therefore, ctDNA analysis can be a useful tool for monitoring PDAC treatment response. These results should be further validated in larger sample numbers.

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

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

Ultrasensitive plasma ctDNA KRAS assay for detection, prognosis, and assessment of therapeutic response in patients with unresectable pancreatic ductal adenocarcinoma.

Precision oncology requires sensitive and specific clinical biomarkers. Carbohydrate Antigen 19-9 (CA19-9) is widely used in pancreatic ductal adenocarcinoma (PDA) but lacks sensitivity and specificity. Nearly all PDAs harbor somatic KRAS mutations, nominating circulating tumor DNA (ctDNA) KRAS as an alternative disease biomarker, however, variable clinical performance has limited its clinical utility. We applied an ultrasensitive, PCR mutation enrichment, next generation sequencing ctDNA KRAS assay in a large cohort of patients with unresectable PDA (N = 189) recruited to the BIOPAC study between 2008–2015. Baseline and longitudinal serum CA19-9 and plasma ctDNA KRAS were correlated with time to progression (TTP) and overall survival (OS). Baseline ctDNA KRAS detection rate was 93.7% (86.4% in patients with non-elevated CA19-9). ctDNA KRAS and CA19-9 were positively correlated yet independently associated with TTP and OS (ctDNA KRAS p = 0.0018 and 0.0014; CA19-9 p = 0.0294 and 0.0007, respectively). A generated model quantitating longitudinal ctDNA KRAS correctly assessed greater than 80% of patient responses. Quantitative detection of KRAS ctDNA is an informative prognostic biomarker, complementary to CA19-9 in patients with unresectable PDA. Longitudinal ctDNA KRAS may inform therapeutic decision making and provides a kinetically dynamic and quantitative metric of patient response.

Detecting KRAS and NRAS resistance mutations in plasma of lung cancer patients.

e20511 Background: Mutations in KRAS and NRAS have been found in a number of malignancies, particularly metastatic colorectal cancer, lung adenocarcinoma, and thyroid cancer. Several studies have shown that specific mutations in KRAS and NRAS are less likely to respond to anti-EGFR therapies. Several diagnostics have been developed to identify such mutations. However, in a large number of cancer patients, tumor biopsy poses a significant health risk thereby limiting the tissue available for current molecular diagnostics. Non-invasive liquid biopsy diagnostics are revolutionizing cancer treatment selection, patient prognosis, and monitoring. Low abundance circulating tumor DNA (ctDNA) is purified from patient plasma and used as an alternative source of genomic material for detecting tumor mutations. Currently available qPCR kits for detecting RAS mutations have a limited sensitivity and coverage since they are not optimized for liquid biopsy purposes. Methods: We have developed a two-step PCR to qPCR method to identify down to single mutant copies of KRAS or NRAS in ctDNA isolated from plasma. The mutant copies are first enriched by a novel multiplexed formula of primers and wildtype (WT) allele blockers. The mutations are then identified by EntroGen’s RAS Mutation Screening Panel or next generation sequencing (NGS). Synthetic mutant DNA was spiked in a background of WT DNA purified from plasma to yield a range of mutant allelic burden over WT background. The dilutions were analyzed to determine the method’s sensitivity. We subsequently used the method to screen 53 non-small cell lung cancer (NSCLC) patient samples. Results: Our two-step method detects single digit mutant copies of 35 clinically significant KRAS and NRAS mutations. Furthermore, NGS experiments demonstrated the two-step method can enrich mutations more than 52-fold. KRAS 12/13 mutations were identified in 6 out of 53 (11%) NSCLC patients. Conclusions: ctDNA-based liquid biopsy tests have been employed by few laboratories but are generally available only as a service. EntroGen’s platform agnostic liquid biopsy RAS assay provides clinicians the tools necessary to determine cancer treatment and monitor patients using most PCR and qPCR instruments.

Abstract A20: Detection and quantification of ctDNA KRAS mutations from patients with unresectable pancreatic cancer

Abstract Introduction: An estimated 90% of pancreatic cancers harbor somatic KRAS G12/G13 mutations. The presence of circulating tumor DNA (ctDNA) KRAS mutations at diagnosis has prognostic implications, however, recent studies on small numbers of patients demonstrate widely variable KRAS ctDNA sensitivity (27 – 71%). Accurate identification of both the presence of ctDNA KRAS mutations and quantification of the number of ctDNA KRAS mutant copies would be an improved therapeutic response biomarker over CA19-9 which is known to be uninformative in 5-10% of patients with pancreatic cancer. A diagnostic and prognostic tool with very high analytical and clinical sensitivity, along with quantification, is needed for therapeutic response monitoring. Patients and Methods: Pretreatment (baseline) and longitudinal plasma samples were prospectively collected from 239 patients with unresectable pancreatic cancer through the Danish Biomarkers in Patients with Pancreatic Cancer (BIOPAC) study. The median age at diagnosis was 67 years (range 42 – 89 years) and 51.9% (N = 112) of patients were male. The majority of patients were metastatic at diagnosis (79.6%; N=172). ctDNA KRAS G12A/C/D/R/S/V, and G13D mutations were PCR enriched, sequenced by next generation sequencing (NGS), quantified and standardized. This was achieved by generating standard curves from a sample set with known numbers of spike-in copies for mutant KRAS molecules. These sample sets were assayed in parallel with patient samples starting with PCR enrichment of mutant KRAS DNA followed by NGS. The number of mutant copies detected was standardized by normalizing the number of copies detected in the sample to a constant number of calculated genome equivalents (GEqs) of wild type DNA across all samples evaluated. Baseline KRAS mutant levels were assessed. Additional metrics analyzed include age, gender, stage, and CA19-9 levels. Results: Two-hundred and sixteen patients (90.4%) had baseline plasma samples which passed quality control metrics. Baseline serum CA19-9 levels were available for 160 patients and ranged from 2.5 – 608,500 U/mL (median 202 U/mL). Eighteen patients (11.3%) had serum CA19-9 levels below the diagnostic threshold (&amp;lt;37 U/mL), and 76 patients (47.5%) had serum CA19-9 levels below the prognostic threshold (&amp;lt;1000 U/mL). Overall, 87% of patients were positive for a KRAS mutation at baseline by ctDNA analysis, with a median of 88.51 copies per 100,000 GEqs. Patients presenting with metastatic disease had a significantly higher median number of KRAS mutations (104.2 versus 23.9 copies per 100,000 GEqs; p&amp;lt;0.0001) and serum CA19-9 levels (1702.0 versus 303.0 U/mL; p&amp;lt;0.0001) at baseline than those with locally advanced cancer. Conclusion: In this large, prospective dataset of 216 patients with unresectable pancreatic cancer, ctDNA analysis was KRAS positive in 87.0% of patients. This detection rate closely matches the published prevalence of KRAS in pancreatic cancer (90%), and out performs previous studies, demonstrating the superior assay sensitivity. ctDNA analysis offers a viable tissue biopsy alternative for determining KRAS mutation status, especially in late stage patients. Given that approximately 48% of patients had CA19-9 levels below the prognostic threshold, quantification of KRAS mutant copy load may provide a more informative biomarker. Other clinical variables, baseline and longitudinal KRAS mutant levels, and patient outcomes will be examined and presented. Citation Format: Inna Chen, Victoria M. Raymond, Jennifer A. Geis, Sandeep Pingle, Eric A. Collisson, Vlada Melnikova, Mark G. Erlander, Julia A. Johansen, Fernando Blanco.{Authors}. Detection and quantification of ctDNA KRAS mutations from patients with unresectable pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A20.