Beyond basics: Key mutation selection features for successful tumor-informed ctDNA detection.

Tumor-informed mutation-based approaches are frequently used for detection of circulating tumor DNA (ctDNA). Not all mutations make equally effective ctDNA markers. The objective was to explore if prioritizing mutations using mutational features-such as cancer cell fraction (CCF), multiplicity, and error rate-would improve the success rate of tumor-informed ctDNA analysis. Additionally, we aimed to develop a practical and easily implementable analysis pipeline for identifying and prioritizing candidate mutations from whole-exome sequencing (WES) data. We analyzed WES and ctDNA data from three tumor-informed ctDNA studies, one on bladder cancer (Cohort A) and two on colorectal cancer (Cohorts I and N). The studies included 390 patients. For each patient, a unique set of mutations (median mutations/patient: 6, interquartile 13, range: 1-46, total n = 4023) were used as markers of ctDNA. The tool PureCN was used to assess the CCF and multiplicity of each mutation. High-CCF mutations were detected more frequently than low-CCF mutations (Cohort A: odds ratio [OR] 20.6, 95% confidence interval [CI] 5.72-173, p = 1.73e-12; Cohort I: OR 2.24, 95% CI 1.44-3.52, p = 1.66e-04; and Cohort N: OR 1.78, 95% CI 1.14-2.79, p = 7.86e-03). The detection-likelihood was additionally improved by selecting mutations with multiplicity of two or above (Cohort A: OR 1.55, 95% CI 1. 14-2.11, p = 3.85e-03; Cohort I: OR 1.78, 95% CI 1.23-2.56, p = 1.34e-03; and Cohort N: OR 1.94, 95% CI 1.63-2.31, p = 2.83e-14). Furthermore, selecting the mutations for which the ctDNA detection method had the lowest error rates, additionally improved the detection-likelihood, particularly evident when plasma cell-free DNA tumor fractions were below 0.1% (p = 2.1e-07). Selecting mutational markers with high CCF, high multiplicity, and low error rate significantly improve ctDNA detection likelihood. We provide free access to the analysis pipeline enabling others to perform qualified prioritization of mutations for tumor-informed ctDNA analysis.

Circulating tumour DNA and risk of recurrence in patients with asymptomatic versus symptomatic colorectal cancer.

BACKGROUND: Multiple initiatives aim to develop circulating tumour DNA (ctDNA) tests for early cancer detection in asymptomatic individuals. The few studies describing ctDNA-testing in both asymptomatic and symptomatic patients report lower ctDNA detection in the asymptomatic patients. Here, we explore if asymptomatic patients differ from symptomatic patients e.g. by including a 'low-ctDNA-shedding' and 'less-aggressive' subgroup. METHODS: ctDNA assessment was performed in two independent cohorts of consecutively recruited patients with asymptomatic colorectal cancer (CRC) (Cohort#1: n = 215, Cohort#2: n = 368) and symptomatic CRC (Cohort#1: n = 117, Cohort#2: n = 722). RESULTS: After adjusting for tumour stage and size, the odds of ctDNA detection was significantly lower in asymptomatic patients compared to symptomatic patients (Cohort#1: OR: 0.4, 95%CI: 0.2-0.8, Cohort#2: OR: 0.7, 95%CI: 0.5-0.9). Further, the recurrence risk was lower in asymptomatic patients (Cohort#1: sHR: 0.6, 95%CI: 0.3-1.2, Cohort#2: sHR: 0.6, 95%CI: 0.4-1.0). Notably, ctDNA-negative asymptomatic patients had the lowest recurrence risk compared to the symptomatic patients (Cohort#1: sHR: 0.2, 95%CI: 0.1-0.6, Cohort#2: sHR: 0.3, 95%CI: 0.2-0.6). CONCLUSIONS: Our study suggests that asymptomatic patients are enriched for a 'low-ctDNA-shedding-low-recurrence-risk' subgroup. Such insights are needed to guide ctDNA-based early-detection initiatives and should prompt discussions about de-escalation of therapy and follow-up for ctDNA-negative asymptomatic CRC patients.

Error-Corrected Deep Targeted Sequencing of Circulating Cell-Free DNA from Colorectal Cancer Patients for Sensitive Detection of Circulating Tumor DNA.

Circulating tumor DNA (ctDNA) is a promising biomarker, reflecting the presence of tumor cells. Sequencing-based detection of ctDNA at low tumor fractions is challenging due to the crude error rate of sequencing. To mitigate this challenge, we developed ultra-deep mutation-integrated sequencing (UMIseq), a fixed-panel deep targeted sequencing approach, which is universally applicable to all colorectal cancer (CRC) patients. UMIseq features UMI-mediated error correction, the exclusion of mutations related to clonal hematopoiesis, a panel of normal samples for error modeling, and signal integration from single-nucleotide variations, insertions, deletions, and phased mutations. UMIseq was trained and independently validated on pre-operative (pre-OP) plasma from CRC patients (n = 364) and healthy individuals (n = 61). UMIseq displayed an area under the curve surpassing 0.95 for allele frequencies (AFs) down to 0.05%. In the training cohort, the pre-OP detection rate reached 80% at 95% specificity, while it was 70% in the validation cohort. UMIseq enabled the detection of AFs down to 0.004%. To assess the potential for detection of residual disease, 26 post-operative plasma samples from stage III CRC patients were analyzed. From this we found that the detection of ctDNA was associated with recurrence. In conclusion, UMIseq demonstrated robust performance with high sensitivity and specificity, enabling the detection of ctDNA at low allele frequencies.

Error Characterization and Statistical Modeling Improves Circulating Tumor DNA Detection by Droplet Digital PCR.

BACKGROUND: Droplet digital PCR (ddPCR) is a widely used and sensitive application for circulating tumor DNA (ctDNA) detection. As ctDNA is often found in low abundance, methods to separate low-signal readouts from noise are necessary. We aimed to characterize the ddPCR-generated noise and, informed by this, create a sensitive and specific ctDNA caller. METHODS: We built 2 novel complimentary ctDNA calling methods: dynamic limit of blank and concentration and assay-specific tumor load estimator (CASTLE). Both methods are informed by empirically established assay-specific noise profiles. Here, we characterized noise for 70 mutation-detecting ddPCR assays by applying each assay to 95 nonmutated samples. Using these profiles, the performance of the 2 new methods was assessed in a total of 9447 negative/positive reference samples and in 1311 real-life plasma samples from colorectal cancer patients. Lastly, performances were compared to 7 literature-established calling methods. RESULTS: For many assays, noise increased proportionally with the DNA input amount. Assays targeting transition base changes were more error-prone than transversion-targeting assays. Both our calling methods successfully accounted for the additional noise in transition assays and showed consistently high performance regardless of DNA input amount. Calling methods that were not noise-informed performed less well than noise-informed methods. CASTLE was the only calling method providing a statistical estimate of the noise-corrected mutation level and call certainty. CONCLUSIONS: Accurate error modeling is necessary for sensitive and specific ctDNA detection by ddPCR. Accounting for DNA input amounts ensures specific detection regardless of the sample-specific DNA concentration. Our results demonstrate CASTLE as a powerful tool for ctDNA calling using ddPCR.

Total cell-free DNA, carcinoembryonic antigen, and C-reactive protein for assessment of prognosis in patients with metastatic colorectal cancer.

In late-stage metastatic colorectal cancer, difficult treatment decisions should incorporate a thorough evaluation of the patient's general condition and subject for shared decision making. Assessment of the individual patients' prognosis is valuable in this setting. The aim was to analyze the prognostic value of plasma levels of total cell-free DNA, carcinoembryonic antigen and C-reactive protein in 97 heavily pretreated patients with metastatic colorectal cancer. Patients received irinotecan, cetuximab, and everolimus in a phase-2 clinical trial ( clinicaltrials.gov NCT01387880). Plasma samples were used for DNA purification and quantification of total cell-free DNA by droplet digital polymerase chain reaction. Serum carcinoembryonic antigen and C-reactive protein were analyzed by routine methods. Clinical endpoints were overall survival and progression-free survival. A total of 82 patients had blood samples available for quantification of total cell-free DNA. Patients with pre-treatment cell-free DNA levels higher than the median total cell-free DNA (9800 alleles per milliliter plasma) had a significantly shorter overall survival of 4.3 months (95% confidence interval: 3.6-5.8) compared to patients with cell-free DNA levels below the median with an overall survival of 11.3 months (95% confidence interval: 8.0-14.8, p < 0.0001). When using the upper normal limit from a previously analyzed normal control group, the median overall survival was 11.3 (95% confidence interval: 7.3-14.8) and 4.3 (95% confidence interval: 3.7-6.1) months, respectively (p < 0.0001). Serum carcinoembryonic antigen and C-reactive protein had similar prognostic value with short overall survival and progression-free survival in patients with elevated levels compared to those within normal range. A high-risk profile of elevated cell-free DNA, carcinoembryonic antigen, and C-reactive protein was described, but in combined Cox regression multivariate analysis, only total cell-free DNA preserved a strong prognostic value. In conclusion, total cell-free DNA in plasma, carcinoembryonic antigen, and C-reactive protein could all contribute to assessment of patients' prognosis and potentially aid in clinical decision making in patients with metastatic colorectal cancer.

The T790M resistance mutation in EGFR is only found in cfDNA from erlotinib-treated NSCLC patients that harbored an activating EGFR mutation before treatment.

BACKGROUND: Lung cancer patients with an activating mutation in the EGFR (epidermal growth factor receptor) can develop resistance to erlotinib treatment, which is often mediated by the T790M resistance mutation in EGFR. The difficulties in obtaining biopsies at progression make it challenging to investigate the appearance of the T790M mutation at progression in large patient cohorts. We have used cell free DNA (cfDNA) from patients treated with erlotinib to investigate if the development of a T790M mutation coincides with the presence of an activating EGFR mutation in the pre-treatment blood sample. METHODS: A cohort of 227 NSCLC (non-small cell lung cancer) adenocarcinoma patients was treated with erlotinib irrespective of EGFR-mutational status. Blood samples were drawn immediately before erlotinib treatment was initiated and again at progression. The cobas® EGFR Mutation Test v2 designed for cfDNA was used to identify 42 EGFR mutations. RESULTS: Of the 227 NSCLC patients, blood samples were available from 144 patients both before erlotinib treatment and at progression (within 1 month before or after clinical progression). One hundred and twenty-eight of the 144 were wild-type EGFR before treatment, and we demonstrate that the T790M mutation was not present at progression in any of these. In contrast, in the 16 patients with an activating EGFR mutation in the pre-treatment blood sample six patients (38%) were identified with a T790M mutation in the progression blood sample. CONCLUSION: The T790M resistance mutation is only found in the cfDNA of erlotinib-treated NSCLC patients if they have an activating EGFR mutation before treatment.

Correlation between circulating mutant DNA and metabolic tumour burden in advanced non-small cell lung cancer patients.

BACKGROUND: Mutated circulating cell-free DNA (cfDNA) has been suggested as a surrogate marker of tumour burden and aggressiveness of disease. We examined the association between the level of plasma mutant cfDNA and metabolic tumour burden (MTB) measured by 18F-fluoro-D-glucose positron emission tomography/computed tomography (18F-FDG PET/CT). Furthermore, the presence of mutant cfDNA was correlated with patient survival. METHODS: Forty-six advanced non-small cell lung cancer (NSCLC) patients were included. At the time of inclusion, blood sampling and a PET/CT scan were performed. cfDNA was isolated and next-generation sequencing (NGS) was performed (Ion AmpliSeq Colon and Lung Cancer panel v2). MTB was defined by a volumetric PET parameter. RESULTS: NGS succeeded in 41 patients. Mutations were detected in the blood of 24 patients. A significant correlation between the allele frequency of the most frequent mutation and MTB was found (P=0.001). Patients with detectable mutated cfDNA had a significantly shorter median overall survival compared with patients without (3.7 versus 10.6 months, P=0.019). This impact on survival was independent of the MTB. CONCLUSIONS: Level of mutated cfDNA tends to correlate with MTB in advanced-stage NSCLC patients. Patients with detectable mutant DNA in plasma had an inferior survival, indicating that this could be an important predictor of survival.

Ultra-micro samples can be used for mRNA quantification of lung cancer biomarkers.

BACKGROUND: Isolating sufficient material for molecular testing remains challenging in non-small cell lung cancer (NSCLC). The use of new ultra-microsamples (uMS) is proven sufficient for DNA and mRNA detection, but whether uMS are useful for quantifying mRNA expression is unknown. We investigated if uMS from lung cancer patients can be used to generate quantitative data on mRNA expression. METHODS: uMS were collected from primary tumors and lymph nodes from patients suspected of having lung cancer. mRNA was isolated, reverse-transcribed into cDNA and quantified with quantitative PCR assays for hepatocyte growth factor receptor (MET), hepatocyte growth factor (HGF), epidermal growth factor receptor (EGFR) and amphiregulin (AREG) mRNA. The fraction of tumor cells to normal cells was estimated in each sample. RESULTS: MET, HGF, EGFR, and AREG expression were evaluated in 90 samples (30 containing cancer cells and 60 without cancer cells). MET and EGFR expression were negligible in samples without cancer cells. In samples containing cancer cells, MET and EGFR could be quantified in 13 samples each. Adjustment for tumor-cell fraction made it possible to obtain a quantitative result for the tumor-cell mRNA expression of MET and EGFR. In contrast, AREG and HGF were expressed in samples without tumor cells. These samples were used to establish the AREG and HGF mRNA expression in normal cells. Seven out of 14 AR-positive and two out of eight HGF-positive samples with tumor cells were above a cut-off of the mean + 2SD established in samples without tumor cells. CONCLUSION: We demonstrate that uMS contain high-quality mRNA, and quantitative studies can be performed when the tumor-cell fraction is considered.

DREAMS: deep read-level error model for sequencing data applied to low-frequency variant calling and circulating tumor DNA detection.

Circulating tumor DNA detection using next-generation sequencing (NGS) data of plasma DNA is promising for cancer identification and characterization. However, the tumor signal in the blood is often low and difficult to distinguish from errors. We present DREAMS (Deep Read-level Modelling of Sequencing-errors) for estimating error rates of individual read positions. Using DREAMS, we develop statistical methods for variant calling (DREAMS-vc) and cancer detection (DREAMS-cc). For evaluation, we generate deep targeted NGS data of matching tumor and plasma DNA from 85 colorectal cancer patients. The DREAMS approach performs better than state-of-the-art methods for variant calling and cancer detection.

Impact of Whole Genome Doubling on Detection of Circulating Tumor DNA in Colorectal Cancer.

OBJECTIVE: Circulating tumor DNA (ctDNA) is a candidate biomarker of cancer with practice-changing potential in the detection of both early and residual disease. Disease stage and tumor size affect the probability of ctDNA detection, whereas little is known about the influence of other tumor characteristics on ctDNA detection. This study investigates the impact of tumor cell whole-genome doubling (WGD) on the detection of ctDNA in plasma collected preoperatively from newly diagnosed colorectal cancer (CRC) patients. METHODS: WGD was estimated from copy numbers derived from whole-exome sequencing (WES) data of matched tumor and normal DNA from 833 Danish CRC patients. To explore if tumor WGD status impacts ctDNA detection, we applied tumor-informed ctDNA analysis to preoperative plasma samples from all patients. RESULTS: Patients with WGD+ tumors had 53% increased odds of being ctDNA positive (OR = 1.53, 95%CI: 1.12-2.09). After stratification for UICC stage, the association persisted for Stage I (OR = 2.44, 95%CI: 1.22-5.03) and Stage II (OR = 1.76, 95%CI: 1.11-2.81) but not for Stage III (OR = 0.83, 95%CI: 0.44-1.53) patients. CONCLUSION: The presence of WGD significantly increases the probability of detecting ctDNA, particularly for early-stage disease. In patients with more advanced disease, the benefit of WGD on ctDNA detection is less pronounced, consistent with increased DNA shedding from these tumors, making ctDNA detection less dependent on the amount of ctDNA released per tumor cell.

Comparing single-target and multitarget approaches for postoperative circulating tumour DNA detection in stage II-III colorectal cancer patients.

Circulating tumour DNA (ctDNA) detection for postoperative risk stratification in cancer patients has great clinical potential. However, low ctDNA abundances complicates detection. Multitarget (MT) detection strategies have been developed to increase sensitivity. Yet, empirical evidence supporting performance gains of MT vs. single-target (ST) strategies in a postoperative setting is limited. We compared ctDNA detection in 379 paired plasma samples from 112 stage II-III colorectal cancer patients by ST digital PCR and MT sequencing of 16 patient-specific variants. The strategies exhibited good concordance (90%, Cohen's Kappa 0.79), with highly correlated ctDNA quantifications (Pearson r = 0.985). A difference was observed in ctDNA detection preoperatively (ST 72/92, MT 88/92). However, no difference was observed immediately after surgery in recurrence (ST 11/22, MT 10/22) or nonrecurrence (both 2/34) patients. In serial samples, detection was similar within recurrence (ST 13/16, MT 14/16) and nonrecurrence (ST 3/49, MT 1/49) patients. Both approaches yielded similar lead times to standard-of-care radiology (ST 4.0 months, MT 4.1 months). Our findings do not support significant performance gains of the MT strategy over the ST strategy for postoperative ctDNA detection.

cGAS-STING pathway expression as a prognostic tool in NSCLC.

BACKGROUND: Disease recurrence in localized lung adenocarcinoma is a major obstacle for improving the overall outcome of lung cancer. Thus, better prognostic biomarkers are needed to identify patients at risk. In order to clear cancer, immune detection of tumor cells is of vital importance. DNA-leakage into the cytosol and tumor environment is one important tumor-associated danger signal and cGAS is a pivotal DNA-sensor that detects misplaced DNA and initiates an innate immune response. In this study, we investigate the cGAS-STING-pathway expression in tumor tissue and circulating immune cells from lung adenocarcinoma patients in relation to stage of disease and overall survival (OS). METHODS: Gene expression was measured using target specific droplet digital polymerase chain reaction (ddPCR) assays in a cohort of 80 patients with lung adenocarcinoma and 45 patients suspected of lung cancer, but determined to be cancer-free. The expression values were correlated to stage of disease. For further exploration of stage dependent expression, we used a publicly available gene expression data set to stratify patients by stage and correlate gene expression to OS. RESULTS: In both tumor tissue and peripheral blood mononuclear cells (PBMCs) from cancer patients, we observed differential expression of cGAS-STING pathway components compared to cancer-free individuals. Furthermore, cGAS-STING pathway expression was elevated in PBMCs from patients with localized disease (stage I and II) compared to patients with metastatic disease (stage III and IV). Survival analysis based on publicly available gene expression data sets demonstrated a superior OS for patients with localized disease and high levels of cGAS, STING and TBK1. CONCLUSIONS: The expression of the cGAS-STING pathway is stage dependent and high expression is correlated with localized adenocarcinoma. For patients with localized disease, high cGAS, STING and TBK1 expression correlated with improved OS and may be a potential biomarker for this patient subgroup.

Development of blood-based biomarker tests for early detection of colorectal neoplasia: Influence of blood collection timing and handling procedures.

INTRODUCTION: Blood-based, cancer-associated biomarkers are susceptible to a variety of well-known preanalytical factors. The influence of bowel preparation before a diagnostic colonoscopy on biomarker levels is, however, poorly investigated. The present study assessed the influence of bowel preparation on colorectal cancer-associated biomarkers. In addition, the effect of single versus double centrifugation of plasma biomarkers was assessed. METHODS: Blood samples were collected pre- and post-bowel preparation from 125 subjects scheduled for first time diagnostic colonoscopy due to symptoms attributable to CRC. The samples were separated into serum and EDTA plasma, and analyzed by four independent collaborators for: 1) the proteins AFP, CA19-9, CEA, hs-CRP, CyFra21-1, Ferritin, Galectin-3 and TIMP-1, 2) the proteins BAG4, IL6ST, vWF, CD44 and EGFR, 3) the glycoprotein Galectin-3 ligand, and 4) cell-free DNA (cfDNA). Statistical analysis of biomarker data has been performed using mixed modelling, including repeated measures. RESULTS: The biomarkers generally showed negligible variation between pre- and post-bowel preparation except for CyFra21-1, Ferritin, BAG4 and cfDNA. CyFra21-1 levels were systematically reduced with 29% (95% CI 21-36%) by bowel preparation (p ≤ 0.0001). Ferritin was not significantly different between pre- and post-bowel preparation (p = 0.07), however the estimated difference (increase) was 18%. BAG4 was systematically reduced by 12% (95% CI 1-22%, p = 0.04), while cfDNA showed a significant increase of 28% (95% CI 17-39%, p < 0.0001). Double centrifugation compared to single centrifugation showed reduced vWF (ratio 0.86, p ≤ 0.0001) and CD44 (ratio 0.85, p = 0.016), but increased IL6ST levels (ratio 1.18, p = 0.014). CONCLUSIONS: Results of the present study demonstrated systematic, statistically significant differences between pre-bowel and post-bowel preparation levels for three independent blood-based biomarkers (BAG4, CyFra21-1, cfDNA), illustrating the importance of timing of sample collection for biomarker analyses.

Correlation between early dynamics in circulating tumour DNA and outcome from FOLFIRI treatment in metastatic colorectal cancer.

Chemotherapy resistance remains a challenge in the clinical management of metastatic colorectal cancer (mCRC). Here, early changes in cell-free circulating tumour DNA (ctDNA) levels were explored as a marker of therapeutic efficacy. Twenty-four mCRC patients were enrolled and treated with FOLFIRI based first-line therapy. Blood samples collected pre-treatment, at day 7, 14, 21, 60 and at progression were analysed for cell-free DNA (cfDNA) and ctDNA levels using digital droplet PCR. A subset of samples were additionally analysed by targeted sequencing. Patients with high pre-treatment ctDNA or cfDNA levels (≥75th centile) had significantly shorter progression free survival (PFS) than patients with lower levels. Despite an overall decline in ctDNA levels from pre-treatment to first CT-scan, serial analysis identified seven patients with temporary increases in ctDNA consistent with growth of resistant cells. These patients had shorter PFS and shorter overall survival. Targeted sequencing analyses of cfDNA revealed dramatic changes in the clonal composition in response to treatment. Our study suggests that increasing ctDNA levels during the first cycles of first-line FOLFIRI treatment is a predictor of incipient progressive disease and poorer survival. Thus, we demonstrate the importance of monitoring ctDNA levels as early as one week after treatment onset to enable early detection of treatment failure.

A method for treatment monitoring using circulating tumour DNA in cancer patients without targetable mutations.

BACKGROUND: The potentials of circulating tumour DNA (ctDNA) have been studied for non-invasive disease monitoring in patients with targetable mutations. However, the majority of cancer patients harbour no targetable mutations. A workflow including targeted next-generation sequencing (NGS) and droplet digital PCR (ddPCR) could be used for monitoring treatment in these patients. Thus, our aim was to evaluate the workflow for ctDNA monitoring in a cohort of non-small cell lung cancer patients. METHODS: Forty patients were prospectively included. Plasma samples were collected prior to and during treatment. NGS (Ion AmpliSeq Colon and Lung Cancer panel v2) was performed on ctDNA from pre-treatment samples. The identified mutations were monitored by ddPCR in consecutively collected samples. RESULTS: Mutations were detected in 21 patients. The most commonly mutated genes were TP53 (N=20) and KRAS (N=13). Treatment was discontinued due to non-response in 18 patients. In 16 of these, a simultaneous increase in ctDNA concentration was observed. A twofold ctDNA concentration increase confirmed in a second successive sample predicted non-response on the following imaging in 83% of patients (10/12). CONCLUSION: ctDNA monitoring can be used for early detection of non-response in patients without targetable mutations, and therefore could supplement imaging data for treatment monitoring in this subset of patients.

Measuring KRAS 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.

Increased PD-L1 expression in erlotinib-resistant NSCLC cells with MET gene amplification is reversed upon MET-TKI treatment.

INTRODUCTION: Cancer cells can achieve immune evasion by expressing the programmed death receptor 1 ligand (PD-L1) on the cell surface. Blockade of the receptor (PD-1) can avert this evasion. Here we aim at investigating PD-L1 expression in erlotinib-resistant lung cancer cells with MET proto-oncogene (MET) gene amplification. MATERIALS AND METHODS: We employed an erlotinib-resistant NSCLC cell line with MET gene amplification. PD-L1 mRNA (qPCR) and protein (flow cytometry) expression was investigated after treatment with MET and mitogen-activated protein kinase (MAPK) targeting drugs (crizotinib and SCH772984, respectively). RESULTS: We demonstrate that PD-L1 expression is increased in erlotinib-resistant non-small cell lung cancer (NSCLC) cells with MET gene amplification. Targeted inhibition of MET significantly decreases both gene and protein expression of PD-L1. Further, we demonstrate that inhibiting MAPK also results in a significant decrease in PD-L1 expression. Taken together these results show that expression of PD-L1 in the erlotinib-resistant cell line is associated with MET activity, and the downstream MAPK pathway. CONCLUSIONS: Our results demonstrate that PD-L1 expression is increased in erlotinib resistant NSCLC cells with MET gene amplification and that the increase can be averted by targeted inhibition of MET.

Increase in soluble PD-1 is associated with prolonged survival in patients with advanced EGFR-mutated non-small cell lung cancer treated with erlotinib.

OBJECTIVES: The central immune co-inhibitory programmed cell death receptor/ligand 1 (PD-1/PD-L1) pathway plays a key role in tumor immune evasion in non-small cell lung cancer (NSCLC). Soluble PD-1 (sPD-1) can be detected in the blood, and preclinical evidence suggests that sPD-1 blocks PD-1/-L1 interaction and improves anti-tumor immunity. The present study compares the concentration of sPD-1 in the serum of advanced NSCLC patients with Epidermal Growth Factor Receptor (EGFR) mutation prior to erlotinib treatment and at the time of progression and correlates these results to patient outcome. MATERIALS AND METHODS: Blood samples from 38 patients with EGFR-mutated advanced NSCLC treated with erlotinib were analyzed for sPD-1 by sandwich ELISA. EGFR mutational status was assessed in circulating tumor DNA (ctDNA) and tumor biopsies. RESULTS: sPD-1 could be detected in 21% of patients prior to erlotinib treatment, and at disease progression in 37% (p=0.015). An increase in sPD-1 during erlotinib therapy was found in 34%, a decrease in 8% and no change in 58% of patients. An increase in sPD-1 during treatment was associated with prolonged progression-free (adjusted HR 0.32, p=0.013) and overall survival (adjusted HR 0.33, p=0.006), but not associated with the emergence of EGFR T790M mutation in ctDNA at progression or any clinicopathological factors. CONCLUSION: Patients with an increase in sPD-1 during erlotinib treatment have a more favorable outcome. Our results emphasize the vast clinical impact of the PD-1/PD-L1 axis, and support the existing preclinical evidence in the bioactive function of sPD-1.

The role of epithelial to mesenchymal transition in resistance to epidermal growth factor receptor tyrosine kinase inhibitors in non-small cell lung cancer.

Inhibition of the epidermal growth factor receptor (EGFR) is an important strategy when treating non-small cell lung cancer (NSCLC) patients. However, intrinsic resistance or development of resistance during the course of treatment constitutes a major challenge. The knowledge on EGFR-directed tyrosine kinase inhibitors (TKIs) and their biological effect keeps increasing. Within the group of patients with EGFR mutations some benefit to a much higher degree than others, and for patients lacking EGFR mutations a subset experience an effect. Up to 70% of patients with EGFR mutations and 10-20% of patients without EGFR mutations initially respond to the EGFR-TKI erlotinib, but there is a severe absence of good prognostic markers. Despite initial effect, all patients acquire resistance to EGFR-TKIs. Multiple mechanisms have implications in resistance development, but much is still to be explored. Epithelial to mesenchymal transition (EMT) is a transcriptionally regulated phenotypic shift rendering cells more invasive and migratory. Within the EMT process lays a need for external or internal stimuli to give rise to changes in central signaling pathways. Expression of mesenchymal markers correlates to a bad prognosis and an inferior response to EGFR-TKIs in NSCLC due to the contribution to a resistant phenotype. A deeper understanding of the role of EMT in NSCLC and especially in EGFR-TKI resistance-development constitute one opportunity to improve the benefit of TKI treatment for the individual patient. Many scientific studies have linked the EMT process to EGFR-TKI resistance in NSCLC and our aim is to review the role of EMT in both intrinsic and acquired resistance to EGFR-TKIs.

Dasatinib and Doxorubicin Treatment of Sarcoma Initiating Cells: A Possible New Treatment Strategy.

Background. One of the major challenges affecting sarcoma treatment outcome, particularly that of metastatic disease, is resistance to chemotherapy. Cancer-initiating cells are considered a major contributor to this resistance. Methods. An immortalised nontransformed human stromal (mesenchymal) stem cell line hMSC-TERT4 and a transformed cell line hMSC-TERT20-CE8, known to form sarcoma-like tumours when implanted in immune-deficient mice, were used as models. Receptor tyrosine kinase (RTK) activation was analysed by RTK arrays and cellular viability after tyrosine kinases inhibitor (TKI) treatment with or without doxorubicin was assessed by MTS assay. Results. Initial results showed that the hMSC-TERT4 was more doxorubicin-sensitive while hMSC-TERT20-CE8 was less doxorubicin-sensitive evidenced by monitoring cell viability in the presence of doxorubicin at different doses. The epidermal growth factor receptor (EGFR) was activated in both cell lines. However hMSC-TERT20-CE8 exhibited significantly higher expression of the EGFR ligands. EGFR inhibitors such as erlotinib and afatinib alone or in combination with doxorubicin failed to further decrease cell viability of hMSC-TERT20-CE8. However, inhibition with the TKI dasatinib in combination with doxorubicin decreased cell viability of the hMSC-TERT20-CE8 cell line. Conclusion. Our results demonstrate that dasatinib, but not EGFR-directed treatment, can decrease cell viability of stromal cancer stem cells less sensitive to doxorubicin.