We investigate the current knowledge on circulating tumour DNA (ctDNA) and its clinical utility in predicting outcomes in patients with metastatic colorectal cancer (mCRC).
PubMed, Embase, Cochrane ...Database of Systematic Reviews and Cochrane Central Register of Controlled Trials were searched. Last search 16/12/2020. We included studies on patients with mCRC reporting the predictive or prognostic value of ctDNA. We performed separate random-effects meta-analyses to investigate if baseline ctDNA and early changes in ctDNA levels during treatment were associated with survival. The risk of bias was assessed according to the Quality in Prognosis Studies tool.
Seventy-one studies were included with 6930 patients. Twenty-four studies were included in meta-analyses. High baseline ctDNA level was associated with short progression-free survival (PFS) (HR = 2.2; 95% CI 1.8-2.8; n = 509) and overall survival (OS) (HR = 2.4; 95% CI 1.9-3.1; n = 1336). A small or no early decrease in ctDNA levels during treatment was associated with short PFS (HR = 3.0; 95% CI 2.2-4.2; n = 479) and OS (HR = 2.8; 95% CI 2.1-3.9; n = 583). Results on clonal evolution and lead-time were inconsistent. A majority of included studies (n = 50/71) had high risk of bias in at least one domain.
Plasma ctDNA is a strong prognostic biomarker in mCRC. However, true clinical utility is lacking.
Recent research has focused on the utility of cell free DNA (cfDNA) in serum and plasma for clinical application, especially in oncology. The literature holds promise of cfDNA as a valuable tumour ...marker to be used for treatment selection, monitoring and follow-up. The results, however, are diverging due to methodological differences with lack of standardisation and definition of sensitivity. The new biological information has not yet come into routine use.
The present study presents external standardisation by spiking with non-human DNA fragments to control for loss of DNA during sample preparation and measurement. It also suggests a method to control for admixture of DNA from normal lymphocytes by utilizing the unique immunoglobulin gene rearrangement in the B-cells. The results show that this approach improves the quality of the analysis and lowers the risk of falsely increased values. In conclusion we suggest a new method to improve the accuracy of cfDNA measurements easily incorporated in the current technology.
•Control for contaminating lymphocyte DNA•Progress in the accuracy of cfDNA analysis•Suggested approach easily incorporated
Besides being an invaluable marker of clonal disease in chronic myeloproliferative disorders (CMPDs), the JAK2 V617F mutation and the mutated allele burden have an impact on disease phenotype and may ...provide information on prognosis. Recently, hydroxyurea (HU) has been shown to induce a rapid decline in the JAK2 V617F allele burden. The aim of the present study was to assess the dynamics of the JAK2 V617F allele burden during long-term treatment with HU in a series of patients with CMPDs. The JAK2 V617F allele burden was determined by quantitative PCR in 24 patients of whom 17 received HU, four received anagrelide and three were followed without any cytoreductive therapy. During a median follow-up of 24·2 months, no significant reductions in the JAK2 V617F allele burden were seen in patients treated with HU. We conclude that HU has only a limited effect on the JAK2 V617F allele burden in CMPD.
The purpose of this study was to quantify the free-circulating plasma HER-2 DNA (cfHER-2 DNA) and to assess the ability of analysis to discriminate between patients with primary breast cancer and ...healthy controls in order to detect metastatic recurrence in comparison with serum HER-2 protein and also HER-2 gene amplification.
The study population consisted of 100 patients with primary breast cancer and 50 healthy female donors. An additional 22 patients with metastases were subsequently included. cfHER-2 DNA was quantified with a quantitative PCR method together with a reference gene.
Results: Using a cut-off of 2.5 for the ratio of the cfHER-2 DNA/reference gene, three (of 15) tissue HER-2-positive patients had a ratio >2.5 prior to the detection of metastatic disease. In the post-metastatic/pre-chemotherapy setting, 11 (of 23) tissue HER-2-positive patients with metastases had a ratio >2.5.
There was no difference between absolute preoperative cfHER-2 DNA values for patients with primary breast cancer and those for healthy controls. There was no difference between cfHER-2 DNA values taken within nine months of development of the metastatic disease and the levels in patients without metastases, but there was a significant difference in the corresponding serum HER-2 protein levels in the tissue HER-2-positive patient group.
Conclusion: Amplified HER-2 DNA can be detected in plasma when using a ratio between cfHER-2 DNA and a reference gene. cfHER-2 DNA could not be used to discriminate between patients with primary breast cancer and healthy controls, and could not predict the development of metastatic disease.
Summary
The JAK2 V617F mutation is a frequent genetic event in the three classical Philadelphia‐chromosome negative chronic myeloproliferative disorders (Phneg.‐CMPD), polycythemia vera (PV), ...essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF). Its occurrence varies in frequency in regards to phenotype. The mutation is found in the majority of patients with PV and about half of the patients with ET and IMF. These diseases are clonal stem cell disorders arising in an early stem cell progenitor. The level in the stem cell hierarchy on which the initiating genetic events and the JAK2 V617F mutation occurs is not known. The mutation has so far been detected in all cells of the myeloid lineage, whereas the potential clonal involvement of the lymphoid lineage is controversial. In this study, we detected the JAK2 V617F mutation by real‐time quantitative PCR (qPCR) in both B‐lymphocytes and T‐lymphocytes in a subgroup of patients with Phneg.‐CMPDs. These results demonstrate the origin of the JAK2 V617F positive disorders in an early stem cell with both lymphoid and myeloid differentiation potential.
Abstract Background Lung cancer is one of the most common malignant diseases worldwide and associated with considerable morbidity and mortality. New agents targeting the epidermal growth factor ...system are emerging, but only a subgroup of the patients will benefit from the therapy. Cell free DNA (cfDNA) in the blood allows for tumour specific analyses, including KRAS-mutations, and the aim of the study was to investigate the possible prognostic value of plasma mutated KRAS (pmKRAS) in patients with non-small cell lung cancer (NSCLC). Material and methods Patients with newly diagnosed, advanced NSCLC eligible for chemotherapy were enrolled in a prospective biomarker trial. A pre-treatment blood sample was drawn and subsequently DNA was extracted and pmKRAS analysed. The patients received carboplatin (AUC5) i.v. day 1 and vinorelbine (30 mg/m2 i.v. day 1 and 60 mg/m2 p.o. day 8) for a maximum of six cycles. Response to chemotherapy was evaluated according to RECIST v.1.0 by CT scans of the chest and upper abdomen. The presence of pmKRAS at baseline was assessed by an in-house qPCR method. The primary endpoint was overall survival (OS). Secondary end-points were progression free survival (PFS) and overall response rate. Results The study included 246 patients receiving a minimum of 1 treatment cycle, and all but four were evaluable for response according to RECIST. Forty-three patients (17.5%) presented with a KRAS mutation. OS was 8.9 months and PFS by intention to treat 5.4 months. Patients with a detectable plasma-KRAS mutation had a significantly shorter OS and PFS compared to the wild type (WT) patients (median OS 4.8 months versus 9.5 months, HR 1.87, 95% CI 1.23–2.84, p = 0.0002 and median PFS 3.0 months versus 5.6 months, HR 1.60, 95% CI 1.09–2.37, p = 0.0043). A multivariate Cox regression analysis confirmed the independent prognostic value of pmKRAS in OS but not in PFS. The response rate to chemotherapy was significantly lower in the group of patients with a mutation compared to WT ( p < 0.0001). Conclusion The presence of KRAS mutations in plasma may be a marker of poor prognosis and may also hold predictive value. Further validation in an independent cohort is highly needed.
Local treatment of liver and/or lung metastases from colorectal cancer (CRC) is increasingly used in daily practice and comprises resection, radiofrequency ablation (RFA) and stereotactic ...radiotherapy (SBRT). The need for prognostic markers for patients undergoing such treatment is currently unmet. We investigated post-treatment circulating tumor-specific DNA (ctDNA) analysis and address a possible prognostic value in a pilot study.
From July 2015 to September 2017, patients undergoing standard of care local treatment of liver and/or lung metastases were included in a prospective translational study. Blood samples were drawn 2 weeks after local treatment and during follow-up. CtDNA was detected by ddPCR and a mass spectrometry-based platform MassARRAY
®
.
Post treatment blood samples were available for 35 patients including five with detectable ctDNA (KRAS mutation, n = 2; NRAS mutation, n = 2; BRAF mutation, n = 1) by ddPCR. 17 out of 35 patients (49%) developed recurrence within a median of 273 days (95%CI 95-NA) among patients positive for ctDNA, while the median time to recurrence was not reached for the group of patients negative for ctDNA (p = .03).
The presence of ctDNA following local treatment of metastatic CRC is associated with an increased risk of recurrence and a short time to failure.
Circulating tumor DNA is being extensively investigated as a clinically relevant cancer marker. KRAS mutations are present in 40% of colorectal tumors and monitoring the mutational status together ...with the level of mutated DNA is of great interest. The measurement of DNA from plasma or serum, however, presents a number of challenges that require attention. The amount of DNA is low and highly fragmented and analyses need to be optimized accordingly.
KRAS ARMS-qPCR assays with amplicon lengths of 120 and 85 base pairs, respectively, were compared using positive control material (PCR fragments) and plasma samples from 46 colorectal cancer patients known to harbor a tumor KRAS mutation.
KRAS mutated DNA was detected in significantly more clinical samples using the short amplicon assays compared to the long amplicon assays (74% vs. 61%, p=0.03). The level of mutated DNA in plasma was on average three times higher using short amplicon assays.
Our results reflect the importance of minimizing the assay length when analyzing highly fragmented DNA, especially if these analyses are to be used for treatment monitoring and relapse detection.
•Circulating cell-free DNA is highly fragmented and requires optimized qPCR assays.•The effect of shortening qPCR amplicons for detecting KRAS mutations was measured.•Significantly more samples were found positive using assays with short amplicons.•The level of detected DNA was three times higher with the optimized assays.
Objective
Molecular monitoring of treatment response in patients with chronic myelogenous leukemia is performed using the Europe Against Cancer (EAC) qPCR assay using the International Scale (IS). ...The assay amplifies both e13a2 and e14a2 BCR‐ABL1 transcript variants. Observing distinct variant‐dependent amplification curves during qPCR, we aimed to determine if this affected quantitation of BCR‐ABL1.
Methods
We investigated the qPCR efficiency at three Danish diagnostic centers (Zealand University Hospital ZUH, Aarhus University Hospital AU, and Rigshospitalet RH) on cell lines expressing either the e13a2 or e14a2 BCR‐ABL1 transcript variants and compared %IS values from 219 chronic myeloid leukemia patients from the centers with either the e13a2 (n = 113) or e14a2 (n = 106) transcript variants obtained by qPCR with absolute quantitation by droplet digital PCR (ddPCR).
Results
Although no significant differences were found in amplification efficiencies of the transcript variants, Bland‐Altman analysis of qPCR vs ddPCR values for patient samples revealed a significant average difference in the bias between variants (e3a2/e14a2) of 4.6‐, 6.5‐, and 1.8‐fold for ZUH, AU, and RH, respectively. Furthermore, qPCR %IS values of diagnostic patient samples revealed a significant 4.7‐fold difference between the e13a2 and e14a2 variants.
Conclusion
Our findings suggest that the EAC qPCR assay may underestimate the e14a2 variant compared to the e13a2 variant.
BACKGROUNDPatients with detectable ctDNA after radical-intent treatment of metastatic spread from colorectal cancer (mCRC) have a very high risk of recurrence, which may be prevented with intensified ...adjuvant chemotherapy (aCTh). In the OPTIMISE study, we investigate ctDNA-guided aCTh after radical-intent treatment of mCRC. Here we present results from the preplanned interim analysis.MATERIAL AND METHODSThe study is an open-label 1:1 randomized clinical trial comparing ctDNA-guided aCTh against standard of care (SOC), with a run-in phase investigating feasibility measures. Key inclusion criteria; radical-intent treatment for mCRC and clinically eligible for triple-agent chemotherapy. Patients underwent a PET-CT scan before randomization. ctDNA analyses of plasma samples were done by ddPCR, detecting CRC-specific mutations and methylation of the NPY gene. In the ctDNA-guided arm, ctDNA positivity led to an escalation strategy with triple-agent chemotherapy, and conversely ctDNA negativity led to a de-escalation strategy by shared-decision making. Patients randomized to the standard arm were treated according to SOC. Feasibility measures for the run-in phase were; the inclusion of 30 patients over 12 months in two Danish hospitals, compliance with randomization >80%, rate of PET-CT-positive findings <20%, and eligibility for triple-agent chemotherapy >80%.RESULTSThirty-two patients were included. The rate of PET-CT-positive cases was 22% (n = 7/32). Ninety-seven percent of the patients were randomized. Fourteen patients were randomly assigned to SOC and sixteen to ctDNA-guided adjuvant treatment and follow-up. All analyses of baseline plasma samples in the ctDNA-guided arm passed the quality control, and 19% were ctDNA positive. The median time to result was three working days. All ctDNA-positive patients were eligible for triple-agent chemotherapy.CONCLUSIONThe study was proven to be feasible and continues in the planned large-scale phase II trial. Results from the OPTIMISE study will potentially optimize the adjuvant treatment of patients undergoing radical-intent treatment of mCRC, thereby improving survival and reducing chemotherapy-related toxicity.
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