Quantitative cell-free DNA, KRAS, and BRAF mutations in plasma from patients with metastatic colorectal cancer during treatment with cetuximab and irinotecan

Abstract Purpose: The present study investigated the levels of circulating cell-free DNA (cfDNA) in plasma from patients with metastatic colorectal cancer (mCRC) in relation to third-line treatment with cetuximab and irinotecan and the quantitative relationship of cfDNA with tumor-specific mutations in plasma. Experimental Design: Inclusion criteria were histopathologically verified chemotherapy-resistant mCRC, adequate Eastern Cooperative Oncology Group performance status, and organ function. Treatment consisted of irinotecan being administered at 350 mg/m2 for 3 weeks and weekly administration of 250 mg/m2 cetuximab until progression or unacceptable toxicity. A quantitative PCR method was developed to assess the number of cfDNA alleles and KRAS and BRAF mutation alleles in plasma at baseline. Results: The study included 108 patients. Only three patients were positive for BRAF mutations. The majority of KRAS mutations detected in tumors were also found in the plasma [32 of 41 (78%)]. Plasma cfDNA and plasma mutant KRAS levels (pmKRAS) were strongly correlated (r = 0.85, P &amp;lt; 10−4). The disease control rate was 77% in patients with low cfDNA (&amp;lt;25% quartile) and 30% in patients with high cfDNA [&amp;gt;75% quartile (P = 0.009)]. Patients with pmKRAS levels higher than 75% had a disease control rate of 0% compared with 42% in patients with lower pmKRAS (P = 0.048). Cox analysis confirmed the prognostic importance of both cfDNA and pmKRAS. High levels were clear indicators of a poor outcome. Conclusions: KRAS analysis in plasma is a viable alternative to tissue analysis. Quantitative levels of cfDNA and pmKRAS are strongly correlated and hold promise of clinical application. Clin Cancer Res; 18(4); 1177–85. ©2012 AACR.</jats:p

Pemetrexed and Gemcitabine for Chemotherapy Refractory Colorectal Cancer-Results of a Phase II and Translational Research Study

ABSTRACT Introduction: We investigated the safety and efficacy of pemetrexed with gemcitabine in heavily pre-treated, chemotherapy refractory, KRAS mutated colorectal cancer (mCRC) and the prognostic value of quantitative levels of cell free DNA (cfDNA) in plasma. Methods: Inclusion criteria comprised; histopathologically verified, KRAS mutant, chemotherapy resistant mCRC, adequate organ function and performance status. Patients received pemetrexed (initially 500 mg/m 2 q3w) + gemcitabine (1250 mg/m 2 days 1 and 8) until progression or unacceptable toxicity. RECIST version 1.1, NCI-CTCAE version 4.0 and Kaplan-Meier statistics were used for endpoint evaluation. Cell free DNA was quantified from pre-treatment EDTA plasma-samples by an in-house qPCR. Results: Forty patients were included. The median number of cycles was 3 (range 0 -12). Thirty-six percent obtained disease stabilisation, but no objective response was observed. Median PFS and OS were 2.8 (range 2.1 -4.0) and 5.4 (range 4.3 -7.0) months, respectively. Adverse events caused immediate discontinuation of treatment or delay of the next cycle and consequently discontinuation in 5 patients. Translational research revealed a shorter PFS and OS with increasing levels of cfDNA. The median PFS in patients with cfDNA levels above the 75 percentile was 2 months compared to 4 months in the remaining patients, HR 3.23 (1.05 -9.89), p = 0.0008. The median OS was 3 and 6 months, respectively, HR 2.9 (95%CI 0. 98 -8.34). Cox regression analysis confirmed that cfDNA remained a significantly independent prognostic factor for both PFS and OS. Conclusion: Pemetrexed and gemcitabine did not prove sufficient benefit and unacceptable toxicity was observed. The potential value of cfDNA should be investigated further

The Importance of Feasibility Assessment in the Design of ctDNA Guided Trials – Results From the OPTIPAL II Study

Introduction: Both quantitative and molecular changes in ctDNA can hold important information when treating metastatic colorectal cancer (mCRC), but its clinical utility is yet to be established. Before conducting a large-scale randomized trial, it is essential to test feasibility. This study investigates whether ctDNA is feasible for detecting patients who will benefit from treatment with epidermal growth factor receptor inhibitors and the prognostic value of circulating tumor DNA (ctDNA) response. Materials and methods: Patients with mCRC, who were considered for systemic palliative treatment and were eligible for ctDNA analysis. Mutational testing on cell-free DNA (cfDNA) was done by ddPCR. ctDNA response from baseline to the third treatment cycle was evaluated in patients with detectable ctDNA at baseline. ctDNA maximum response was defined as undetectable ctDNA at the third treatment cycle, ctDNA partial response as any decrease in the ctDNA level, and ctDNA progression as any increase in the ctDNA level. Results: Forty-nine patients were included. The time to test results for mutational testing on cfDNA was significantly shorter than on tumor tissue (p &lt; .001). Progression-free survival were 11.2 months (reference group), 7.5 months (HR = 10.7, p= .02), and 4.6 months (HR = 11.4, p= .02) in patients with ctDNA maximum response, partial response, and progression, respectively. Overall survival was 31.2 months (reference group), 15.2 months (HR = 4.1, p= .03), and 9.0 months (HR = 2.6, p= .03) in patients with ctDNA maximum response, partial response, and progression, respectively. Conclusion: Pretreatment mutational testing on cfDNA in daily clinic is feasible and can be applied in randomized clinical trials evaluating the clinical utility of ctDNA. Early dynamics in ctDNA during systemic treatment hold prognostic value.</p

Pemetrexed and Gemcitabine for Chemotherapy Refractory Colorectal Cancer-Results of a Phase II and Translational Research Study *

ABSTRACT Introduction: We investigated the safety and efficacy of pemetrexed with gemcitabine in heavily pre-treated, chemotherapy refractory, KRAS mutated colorectal cancer (mCRC) and the prognostic value of quantitative levels of cell free DNA (cfDNA) in plasma. Methods: Inclusion criteria comprised; histopathologically verified, KRAS mutant, chemotherapy resistant mCRC, adequate organ function and performance status. Patients received pemetrexed (initially 500 mg/m 2 q3w) + gemcitabine (1250 mg/m 2 days 1 and 8) until progression or unacceptable toxicity. RECIST version 1.1, NCI-CTCAE version 4.0 and Kaplan-Meier statistics were used for endpoint evaluation. Cell free DNA was quantified from pre-treatment EDTA plasma-samples by an in-house qPCR. Results: Forty patients were included. The median number of cycles was 3 (range 0 -12). Thirty-six percent obtained disease stabilisation, but no objective response was observed. Median PFS and OS were 2.8 (range 2.1 -4.0) and 5.4 (range 4.3 -7.0) months, respectively. Adverse events caused immediate discontinuation of treatment or delay of the next cycle and consequently discontinuation in 5 patients. Translational research revealed a shorter PFS and OS with increasing levels of cfDNA. The median PFS in patients with cfDNA levels above the 75 percentile was 2 months compared to 4 months in the remaining patients, HR 3.23 (1.05 -9.89), p = 0.0008. The median OS was 3 and 6 months, respectively, HR 2.9 (95%CI 0. 98 -8.34). Cox regression analysis confirmed that cfDNA remained a significantly independent prognostic factor for both PFS and OS. Conclusion: Pemetrexed and gemcitabine did not prove sufficient benefit and unacceptable toxicity was observed. The potential value of cfDNA should be investigated further

Measuring <i>KRAS </i>Mutations in Circulating Tumor DNA by Droplet Digital PCR and Next-Generation Sequencing

Measuring total cell-free DNA (cfDNA) or cancer-specific mutations herein has presented as new tools in aiding the treatment of cancer patients. Studies show that total cfDNA bears prognostic value in metastatic colorectal cancer (mCRC) and that measuring cancer-specific mutations could supplement biopsies. However, limited information is available on the performance of different methods. Blood samples from 28 patients with mCRC and known KRAS mutation status were included. cfDNA was extracted and quantified with droplet digital polymerase chain reaction (ddPCR) measuring Beta-2 Microglobulin. KRAS mutation detection was performed using ddPCR (Bio-Rad) and next-generation sequencing (NGS, Ion Torrent PGM). Comparing KRAS mutation status in plasma and tissue revealed concordance rates of 79% and 89% for NGS and ddPCR. Strong correlation between the methods was observed. Most KRAS mutations were also detectable in 10-fold diluted samples using the ddPCR. We find that for detection of KRAS mutations in ctDNA ddPCR was superior to NGS both in analysis success rate and concordance to tissue. We further present results indicating that lower amount of plasma may be used for detection of KRAS mutations in mCRC

Elevated microRNA-126 is associated with high vascular endothelial growth factor receptor 2 expression levels and high microvessel density in colorectal cancer

MicroRNAs (miRNAs) are involved in a number of biological processes, including tumour biology. Pre-clinical studies have shown that miRNA-126 regulates signalling downstream of vascular endothelial growth factor receptor 2 (VEGFR-2) and, consequently, angiogenesis. The aim of this study was to analyse the possible relationship between miRNA-126, VEGFR-2 and angiogenesis in tumour tissue from patients with colorectal cancer (CRC). Tumour tissue was obtained from 81 patients. The miRNA-126 and VEGFR-2 gene expression levels were analysed by PCR and the protein concentrations of VEGFR-2 were analysed by ELISA. Angiogenesis, visualised by the endothelial cell marker CD105 combined with caldesmon, was assessed by immunohistochemistry and the microvessel density (MVD) technique. In situ hybridisation was performed for miRNA-126. Tumours were classified as low or high miRNA-126-expressing using the median as the cut-off. The median gene expression levels of VEGFR-2 were significantly lower in the tumours expressing low levels of miRNA-126, 0.30 (95% CI, 0.24–0.36), compared to those expressing high levels of miRNA-126, 0.48 (95% CI, 0.28–0.60), p=0.02. A positive association was observed with VEGFR-2 protein concentrations, p=0.06. The median MVD was significantly lower in the tumours expressing low levels of miRNA-126, 5.8 (95% CI, 5.33–6.67), compared to those expressing high levels, 8.0 (95% CI, 6.33–9.00), p<0.01. miRNA-126 was detected in endothelial cells by in situ hybridisation analysis. These results suggest that high levels of miRNA-126 in CRC are associated with high VEGFR-2 mRNA and protein levels and a higher density of newly formed microvessels. However, further studies should be conducted to analyse the clinical value of miRNA-126 in CRC

EGFR related mutational status and association to clinical outcome of third-line cetuximab-irinotecan in metastatic colorectal cancer

<p>Abstract</p> <p>Background</p> <p>As supplement to <it>KRAS </it>mutational analysis<it>, BRAF and PIK3CA </it>mutations as well as expression of PTEN may account for additional non-responders to anti-EGFR-MoAbs treatment. The aim of the present study was to investigate the utility as biomarkers of these mutations in a uniform cohort of patients with metastatic colorectal cancer treated with third-line cetuximab/irinotecan.</p> <p>Methods</p> <p>One-hundred-and-seven patients were prospectively included in the study. Mutational analyses of <it>KRAS, BRAF </it>and <it>PIK3CA </it>were performed on DNA from confirmed malignant tissue using commercially available kits. Loss of PTEN and EGFR was assessed by immunohistochemistry.</p> <p>Results</p> <p>DNA was available in 94 patients. The frequency of KRAS, <it>BRAF </it>and <it>PIK3CA </it>mutations were 44%, 3% and 14%, respectively. All were non-responders. EGF receptor status by IHC and loss of PTEN failed to show any clinical importance. <it>KRAS </it>and <it>BRAF </it>were mutually exclusive. Supplementing <it>KRAS </it>analysis with <it>BRAF </it>and <it>PIK3CA </it>indentified additional 11% of non-responders. Patient with any mutation had a high risk of early progression, whereas triple-negative status implied a response rate (RR) of 41% (p < 0.001), a disease control (DC) rate of 73% (p < 001), and a significantly higher PFS of 7.7(5.1-8.6 95%CI) versus 2.3 months (2.1-3.695%CI) (p < 0.000).</p> <p>Conclusion</p> <p>Triple-negative status implied a clear benefit from treatment, and we suggest that patient selection for third-line combination therapy with cetuximab/irinotecan could be based on triple mutational testing.</p