Plasma KRAS Research Articles

Changes in mutational status during third‐line treatment for metastatic colorectal cancer—Results of consecutive measurement of cell free DNA, KRAS and BRAF in the plasma

KRAS and BRAF mutations are responsible for primary resistance to epidermal growth factor receptor (EGFR) MoAbs in metastatic colorectal cancer (mCRC), but it is unknown what causes wildtype (wt) patients to develop resistance during treatment. We measured circulating free DNA (cfDNA), KRAS and BRAF in plasma and report the changes during third line treatment with cetuximab and irinotecan. One-hundred-and-eight patients received irinotecan 350 mg/m2 q3w and weekly cetuximab (250 mg/m2) until progression (RECIST) or unacceptable toxicity. cfDNA and number of mutated KRAS/BRAF alleles in plasma at baseline and before each cycle was analyzed by an in-house qPCR. cfDNA and pKRAS levels decreased from baseline to cycle three and increased at time of progression (p = 0.008). The decrease was larger in responding patients than in non-responding (p < 0.05). Two patients with primary mutant disease had different types of mutations detected in the plasma, including synchronous KRAS and BRAF. Twelve patients had a primary KRAS mutant tumor, but wild-type disease according to baseline plasma analysis, eight of these obtained stabilization of disease. In five patients with primary wt disease a mutation appeared in plasma before radiological evidence of progression. Loss of mutations may explain observed benefit of treatment in primary mutant disease, whereas appearance of mutations during therapy may be responsible for acquired resistance in primary wt disease. Benefit from EGFR MoAbs may be influenced by the quantitative level of mutational alleles rather than by mutational status alone, and plasma levels of cfDNA, KRAS and BRAF could be used to monitor patients during treatment.

Levels of cell-free DNA and plasma KRAS during treatment of advanced NSCLC

Non-small cell lung cancer (NSCLC) is one of the most common malignant tumours in the western world and is associated with a poor prognosis. Biomarkers predicting prognosis and therapeutic effects are highly required, and cell-free DNA (cfDNA) may be a feasible option. Genetic mutations can be analysed in plasma and may increase the scientific use of such measurements. In the present study, we investigated: i) the dynamics of cfDNA and plasma mutated KRAS (pmKRAS) during the treatment of patients with advanced NSCLC; and ii) the prognostic value of baseline cfDNA and pmKRAS. Sixty‑nine patients were included in a prospective biomarker trial. Inclusion criteria included advanced NSCLC, candidate for first-line treatment, no previous cancer within the five years prior to this study. Blood samples were drawn at baseline, day 8 and at progression. Analyses of cfDNA and KRAS mutations in plasma were performed using an in-house qPCR assay. Evaluation of the treatment effect and status at follow-up was performed according to RECIST 1.1. The median levels of cfDNA were significantly higher at progression (9,250 alleles/ml) than at baseline (5,450 alleles/ml). Overall survival and progression-free survival were both significantly shorter in patients with high levels of cfDNA (above the 75th percentile) compared to lower levels. Only few patients harboured KRAS mutations in plasma. Two patients had no KRAS mutations in plasma at baseline, but mutations appeared in the subsequent blood samples. High baseline levels of cfDNA indicate a poor prognosis. The level changes during the treatment course with a significant increase at progression, suggesting a possible predictive value of cfDNA. The plasma KRAS status may change during treatment with potential implications for treatment selection.

KRAS-mutated plasma DNA as predictor of outcome from irinotecan monotherapy in metastatic colorectal cancer

Background:We investigated the clinical implications of KRAS and BRAF mutations detected in both archival tumor tissue and plasma cell-free DNA in metastatic colorectal cancer patients treated with irinotecan monotherapy.Methods:Two hundred and eleven patients receiving second-line irinotecan (350 mg m−2 q3w) were included in two independent cohorts. Plasma was obtained from pretreatment EDTA blood-samples. Mutations were detected in archival tumour and plasma with qPCR methods.Results:Mutation status in tumor did not correlate to efficacy in either cohort, whereas none of the patients with mutations detectable in plasma responded to therapy. Response rate and disease control rate in plasma KRAS wt patients were 19 and 66% compared with 0 and 37%, in patients with pKRAS mutations, (P=0.04 and 0.01). Tumor KRAS status was not associated with PFS but with OS in the validation cohort. Plasma BRAF and KRAS demonstrated a strong influence on both PFS and OS. The median OS was 13.0 mo in pKRAS wt patients and 7.8 in pKRAS-mutated, (HR=2.26, P<0.0001). PFS was 4.6 and 2.7 mo, respectively (HR=1,69, P=0.01). Multivariate analysis confirmed the independent prognostic value of pKRAS status but not KRAS tumor status.Conclusion:Tumor KRAS has minor clinical impact, whereas plasma KRAS status seems to hold important predictive and prognostic information.

Phase II trial of temsirolimus alone and in combination with irinotecan for KRAS mutant metastatic colorectal cancer: Outcome and results of KRAS mutational analysis in plasma

Background. Patients with chemotherapy refractory metastatic colorectal cancer and KRAS mutations have no effective treatment option. The present study evaluated the efficacy of temsirolimus in chemotherapy refractory mCRC with KRAS mutations. Furthermore, we wanted to investigate if resistance to temsirolimus could be reversed by the addition of irinotecan. Finally, we analyzed pre-treatment blood samples for KRAS mutations to investigate the association between quantitative measures of KRAS mutated alleles and clinical outcome. Material and methods. Patients received weekly temsirolimus 25 mg until progression. Thereafter patients were treated with combination therapy comprising biweekly irinotecan 180 mg/m2 and weekly temsirolimus. A polymerase chain reaction method was used to quantify the KRAS mutated alleles in plasma (pKRAS). Results. Sixty-four patients were included. Treatment was well tolerated. Thirty-eight percent achieved stable disease on monotherapy and 63% on combination therapy. Four and eight patients had a minimal response, respectively. Median overall survival was 160 days. Median time to progression was 45 and 84 days, respectively. The concordance between KRAS status in tumor and plasma was 82%. All patients with tumor reduction had low levels of pKRAS. Patients with high pKRAS had a 77% risk of early progression on monotherapy compared to 43% in patients with lower levels. Multivariate survival analysis confirmed that pKRAS was a strong prognostic factor. Conclusion. Temsirolimus has limited efficacy in chemotherapy resistant KRAS mutant disease, but plasma KRAS quantification is a strong predictor of outcome.

Potential clinical significance of plasma-based KRAS mutation analysis using the COLD-PCR/TaqMan® -MGB probe genotyping method

Despite the improved ability to detect mutations in recent years, tissue specimens cannot always be procured in a clinical setting, particularly from patients with recurrence of tumors or metastasis. Therefore, the aim of this study was to investigate whether plasma is able to be used for mutation analysis instead of tissue specimens. We collected plasma from 62 patients with colorectal cancer (CRC) prior to treatment. DNA extracted from plasma and matched tumor tissues were obtained. Mutations in KRAS were amplified from the tissue specimens and sequenced by regular polymerase chain reaction (PCR) and co-amplification at lower denaturation temperature (COLD)-PCR. Plasma KRAS gene mutation on codon 12 (GGT>GAT) was detected using a nested COLD-PCR/TaqMan(®) -MGB probe. Mutations in plasma and matched tumors were compared. KRAS mutation on codon 12 (GGT>GAT) was found in 13 (21.0%) plasma specimens and 12 (19.4%) matched tumor tissues. The consistency of KRAS mutations between plasma and tumors was 75% (9/12), which indicated a high correlation between the mutations detected in plasma DNA and the mutations detected in the corresponding tumor DNA (P<0.001; correlation index, k=0.649). Notably, four (6.5%) patients with plasma DNA mutations had no detectable KRAS mutations in the corresponding primary tumors, and three (4.8%) patients with tumor DNA mutations had no detectable KRAS mutations in the corresponding plasma DNA samples. Thus, KRAS mutations in plasma DNA correlate with the mutation status in matched tumor tissues of patients with CRC. Our study provides evidence to suggest that plasma DNA may be used as a potential medium for KRAS mutation analysis in CRC using the COLD-PCR/TaqMan-MGB probe method.

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

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

Modified PNA-PCR method

KRAS mutations are proved to confer dramatic resistance to EGFR target therapy of cancer patients. The aim of this study was to establish a convenient and accurate method to screen plasma KRAS mutations of cancer patients since tumor specimens were not always available in clinical practice. A modified PNA-PCR method was established and evaluated in plasma of 19 pancreatic cancer patients. Our results showed that the modified PNA-PCR assay was a sensitive (87.5%, 14/16) and accurate (92.9%, 13/14) method to screen plasma KRAS mutations of pancreatic cancer patients and there was a high consistency of KRAS mutation status between plasma samples and tumor specimens. The modified protocol could not only screen plasma KRAS mutations rapidly and accurately but also had potential to quantify KRAS mutant DNA to predict treatment response of cancer patients and monitor disease progression. It’s should be indicated that this modified assay was only confirmed in the pancreatic cancer patients in this study and need to be verified in other cancer patients.

High-fidelity DNA polymerase-based PNA-PCR plus pyrosequencing: Is it a sensitive and accurate assay for genotyping and quantifying plasma KRAS mutations?

e21006 Background: Plasma DNA may harbor tumor-specific genetic alternations and could be used as surrogate tumor tissues for nonivasive examination of tumor-specific gene mutation. Peptide nucleic acid (PNA)-PCR is reported to be a high sensitivity method to screen KRAS mutation from plasma/serum of cancer patients. However, since DNA polymerase errors could affect the performance of PNA-PCR and most of PNA-PCR reports didn’t take this into consideration, it’s necessary to compare the mutation status between plasma and tumor tissue to evaluate the effect of DNA polymerase errors during PNA-PCR. Methods: High-fidelity DNA polymerase based PNA-PCR plus pyroquencing assay was developed and its detection limit was assessed by dilutions of the KRAS mutant DNA. Using this assay, we investigated KRAS mutations on cell-free DNA from 19 (15 pre-operations and 4 post-operations) pancreatic cancer patients. KRAS mutation status of these 19 patients was also screened on formalin-fixed paraffin-embedded (FFPE) tumor ...

Potential Clinical Significance of a Plasma-Based KRAS Mutation Analysis in Patients with Advanced Non–Small Cell Lung Cancer

Non-small cell lung cancer (NSCLC) with KRAS mutation may be resistant to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI). This study aims to evaluate a plasma-based KRAS mutation analysis and the clinical significance of plasma KRAS mutation as a predictive marker for tumor resistance to EGFR-TKIs in patients with NSCLC. DNA extracted from plasma and matched tumor tissues were obtained from 273 patients with advanced stage NSCLC. Patients were followed up prospectively for treatment outcomes. KRAS mutations in codon 12 and 13 were detected using PCR-restriction fragment length polymorphism. Mutations in plasma and matched tumors were compared. Associations between KRAS mutation status and patients' clinical outcomes were analyzed. KRAS mutation was found in 35 (12.8%) plasma samples and 30 (11.0%) matched tumor tissues. The consistency of KRAS mutations between plasma and tumors is 76.7% (23 of 30; kappa = 0.668; P < 0.001). Among 120 patients who received EGFR-TKI treatment, the response rate was only 5.3% (1 of 19) for patients with plasma KRAS mutation compared with 29.7% for patients with no KRAS mutation in plasma DNA (P = 0.024). The median progression-free survival time of patients with plasma KRAS mutation was 2.5 months compared with 8.8 months for patients with wild-type KRAS (P < 0.001). KRAS mutation in plasma DNA correlates with the mutation status in the matched tumor tissues of patients with NSCLC. Plasma KRAS mutation status is associated with a poor tumor response to EGFR-TKIs in NSCLC patients and may be used as a predictive marker in selecting patients for such treatment.

Results of a Phase I Trial of 12 Patients With Locally Advanced Pancreatic Carcinoma Combining Gefitinib, Paclitaxel, and 3-Dimensional Conformal Radiation

To evaluate the toxicity of daily gefitinib, an epidermal growth factor receptor-tyrosine kinase inhibitor, with concurrent chemoradiation (CRT) in patients with locally advanced pancreatic adenocarcinoma and prospectively evaluate plasma k-ras as a potential marker of response to gefitinib and CRT. Eleven of 12 eligible patients enrolled received a 7-day induction of gefitinib (250 mg PO) followed by daily gefitinib with concurrent CRT. Patients received 50.4 Gy/28 fractions of external beam radiation with weekly paclitaxel (40 mg/m IV) followed by maintenance on gefitinib. Plasma k-ras codon 12 mutations were detected using a two-stage restriction fragment length polymorphism-polymerase chain reaction assay on patients' plasma both before and after therapy. Mutations were confirmed by direct sequencing. Common adverse events included grade 1 skin rash (63%), grade 1 to 2 gastrointestinal symptoms including anorexia, nausea, vomiting, and diarrhea occurred in 63% of patients, grade 3 nausea occurred in 45% of patients. Three patients did not complete therapy, only one was possibly associated with study drug. K-ras mutations were detected in the pre-gefitinib plasma of 5/11 patients and in the matched tumor tissue of 3/4 patients. In patients where k-ras mutations were undetectable post-treatment, survival times were favorable. Combination of daily gefitinib with concurrent CRT in this locally advanced pancreatic cancer population was reasonably tolerated. Rapid changes in serum k-ras may provide critical information as to the efficacy of a novel agent and assist in tailoring treatment for cancers of the pancreas.

6592 POSTER Determination of plasma K-ras mutations in codon 12 in advanced non-small cell lung cancer (NSCLC) patients. Analysis of its prognostic role

6592 POSTER Determination of plasma K-ras mutations in codon 12 in advanced non-small cell lung cancer (NSCLC) patients. Analysis of its prognostic role

PD2-3-1: Prognostic significance and origin of plasma KRAS mutations in patients with non-small cell lung cancer (NSCLC)

PD2-3-1: Prognostic significance and origin of plasma KRAS mutations in patients with non-small cell lung cancer (NSCLC)

Origin and prognostic value of circulating KRAS mutations in lung cancer patients

Because of the current controversy on the origin and clinical value of circulating KRAS codon 12 mutations in lung cancer, we screened 180 patients using a combined restriction fragment-length polymorphism and polymerase chain reaction (RFLP–PCR) assay. We detected KRAS mutations in 9% plasma samples and 0% matched lymphocytes. Plasma KRAS mutations correlated significantly with poor prognosis. We validated the positive results in a second laboratory by DNA sequencing and found matching codon 12 sequences in blood and tumor in 78% evaluable cases. These results support the notion that circulating KRAS mutations originate from tumors and are prognostically relevant in lung cancer.

KRAS mutation analysis in patients (pts) with locally advanced pancreatic cancer (LAPC) treated with gefitinib and chemoradiation therapy (CT-RT) in a phase I trial

4106 Background: Correlative studies that incorporate biomarkers to rapidly analyze response to new agents are needed. Unique to pancreatic cancer is the high incidence of KRAS mutations (over 90%). This pilot study evaluated plasma KRAS mutations for disease monitoring in LAPC pts treated on a Phase I trial combining CT-RT with the EGFR inhibitor, gefitinib. Methods: DNA was extracted from plasma of 11 pts collected at 3 timepoints: pre-gefitinib, pre-CT-RT, and post-gefitinib+CT-RT. Matched tissue DNA was obtained from 4 pts with available paraffin blocks. KRAS codon 12 mutations were detected using a two-stage RFLP-PCR assay. Cell line controls: Calu-1 (mutant KRAS) and LNCaP (wild-type KRAS). Mutations were confirmed by direct DNA sequencing. Results were related to pt clinical data. Results: KRAS mutations were detected in the pre-gefitinib plasma of 5/11 pts, and in the matched tumor tissue of 3/4 pts. Of the 5 pts with plasma KRAS mutations, 2 pts with no detectable mutant KRAS in the plasma post-gefitinib+CT-RT had overall survival of 8 and 21 months, whereas 2 pts who retained mutant KRAS had overall survival of only 2 and 5 months, and one pt withdrew early. Of the 3 tumor tissues containing mutant KRAS, the mutations were also detectable in the matched plasma in 2 pts (67%). KRAS codon 12 mutations spectrum: 4 GGT→GAT, 2 GGT→GTT and 1 GGT→AGT. Conclusions: Plasma KRAS mutations are readily detectable in LAPC pts, and the clearance or persistence of plasma KRAS mutations after treatments reflected the clinical course in some cases. The use of plasma KRAS mutation as a marker of survival and response will be further assessed in a recently approved phase I trial using a proteasome inhibitor with chemoradiation at the University of Colorado. No significant financial relationships to disclose.

K-ras mutations in DNA extracted from the plasma of patients with pancreatic carcinoma: diagnostic utility and prognostic significance.

Previous studies have demonstrated the presence of K-ras mutations in the plasma of patients with pancreatic carcinoma. However, the diagnostic utility and the prognostic significance of this finding have never been addressed. Forty-four consecutive patients with histologically confirmed primary pancreatic ductal adenocarcinoma were included. A control group of 37 patients with chronic pancreatitis, 10 patients with other tumors of the pancreatic area, nine patients with acute pancreatitis, and four healthy volunteers was also included. Plasma DNA was isolated and K-ras codon-12 mutations were analyzed by means of restriction fragment length polymorphism-polymerase chain reaction and single-strand conformation polymorphism techniques. Patients were followed up to establish their clinical outcome. The mutant-type K-ras gene was found in plasma DNA samples of 12 (27%) of 44 patients with pancreatic ductal adenocarcinoma; this finding was related to the tumor stage (P = .05), mainly in the presence of distant metastases (P = .02). In addition, K-ras mutations were detected in the plasma DNA of two (5%) of 37 patients with chronic pancreatitis. In the subset of patients with pancreatic masses, the sensitivity and specificity of plasma K-ras analysis for pancreatic adenocarcinoma were 27% and 100%, respectively. Finally, pancreatic carcinoma patients with the mutant-type K-ras gene in plasma DNA exhibited a shorter survival time than patients with the wild-type gene (P<.005), and plasma K-ras mutations were identified as the only independent prognostic factor (odds ratio, 1.51; 95% confidence interval, 1.02 to 2.23). Plasma K-ras analysis is a highly specific, low-sensitivity approach that has diagnostic and prognostic clinical implications in patients with pancreatic carcinoma.