Functional Contribution and Clinical Implication of Cancer-Associated Fibroblasts in Glioblastoma.

PURPOSE: The abundance and biological contribution of cancer-associated fibroblasts (CAF) in glioblastoma (GBM) are poorly understood. Here, we aim to uncover its molecular signature, cellular roles, and potential tumorigenesis implications. EXPERIMENTAL DESIGN: We first applied single-cell RNA sequencing (RNA-seq) and bioinformatics analysis to identify and characterize stromal cells with CAF transcriptomic features in human GBM tumors. Then, we performed functional enrichment analysis and in vitro assays to investigate their interactions with malignant GBM cells. RESULTS: We found that CAF abundance was low but significantly correlated with tumor grade, poor clinical outcome, and activation of extracellular matrix remodeling using three large cohorts containing bulk RNA-seq data and clinical information. Proteomic analysis of a GBM-derived CAF line and its secretome revealed fibronectin (FN1) as a critical candidate factor mediating CAF functions. This was validated using in vitro cellular models, which demonstrated that CAF-conditioned media and recombinant FN1 could facilitate the migration and invasion of GBM cells. In addition, we showed that CAFs were more abundant in the mesenchymal-like state (or subtype) than in other states of GBMs. Interestingly, cell lines resembling the proneural state responded to the CAF signaling better for the migratory and invasive phenotypes. CONCLUSIONS: Overall, this study characterized the molecular features and functional impacts of CAFs in GBM, alluding to novel cell interactions mediated by CAFs in the GBM microenvironment.

Short-term biological variation of serum tryptase.

OBJECTIVES: Serum tryptase is a biomarker of mast cell activation. Among others, it is used in the diagnosis of anaphylaxis where a significant increase during the acute phase supports the diagnosis. When evaluating changes in biomarker levels, it is of utmost importance to consider the biological variation of the marker. Therefore, the aim of this study was to evaluate the short-term biological variation of serum tryptase. METHODS: Blood samples were drawn at 9 AM three days in a row from apparently healthy subjects. On day two, additional blood samples were drawn every third hour for 12 h. The tryptase concentration was measured in serum using a fluoroenzyme immunoassay (ImmunoCAP™, Thermo Fisher Scientific). Linear mixed-effects models were used to calculate components of biological variation. RESULTS: In 32 subjects, the overall mean concentration of tryptase was 4.0 ng/mL (range, 1.3-8.0 ng/mL). The within-subject variation was 3.7 % (95 % confidence interval (CI) 3.0-4.4 %), the between-subject variation was 31.5 % (95 % CI 23.1-39.8 %), and the analytical variation was 3.4 % (95 % CI 2.9-4.1 %). The reference change value was 13.3 % for an increase in tryptase at a 95 % level of significance. No significant day-to-day variation was observed (p=0.77), while a minute decrease in the serum concentration was observed during the day (p<0.0001). CONCLUSIONS: Serum tryptase is a tightly regulated biomarker with very low within-subject variation, no significant day-to-day variation, and only minor semidiurnal variation. In contrast, a considerable between-subject variation exists. This establishes serum tryptase as a well-suited biomarker for monitoring.

Short-term biological variation of plasma uracil in a Caucasian healthy population.

OBJECTIVES: Plasma uracil is a new biomarker to assess the activity of dihydropyrimidine dehydrogenase before cancer treatment with fluoropyrimidine drugs. Knowledge on the biological variation of plasma uracil is important to assess the applicability of plasma uracil as a biomarker of drug tolerance and efficacy. METHODS: A total of 33 apparently healthy individuals were submitted to sequential blood draws for three days. On the second day, blood draws were performed every third hour for 12 h. Plasma uracil was quantified by LC-MS/MS. The within-subject (CVI) and between-subject (CVG) biological variation estimates were calculated using linear mixed-effects models. RESULTS: The overall median value of plasma uracil was 10.6 ng/mL (range 5.6-23.1 ng/mL). The CVI and CVG were 13.5 and 22.1%, respectively. Plasma uracil remained stable during the day, and there was no day-to-day variation observed. No differences in biological variation components were found between sex and no correlation to age was found. Four samples were calculated to be required to estimate the homeostatic set-point ±15% with 95% confidence. CONCLUSIONS: Plasma uracil is subject to tight homeostatic regulation without semidiurnal and day-to-day variation, however between-subject variation exists. This emphasizes plasma uracil as a well-suited biomarker for evaluation of dihydropyrimidine dehydrogenase activity, but four samples are required to establish the homeostatic set-point in a patient.

Short-term biological variation of serum glial fibrillary acidic protein.

OBJECTIVES: Serum glial fibrillary acidic protein (GFAP) is an emerging biomarker for intracerebral diseases and is approved for clinical use in traumatic brain injury. GFAP is also being investigated for several other applications, where the GFAP changes are not always outstanding. It is thus essential for the interpretation of GFAP to distinguish clinical relevant changes from natural occurring biological variation. This study aimed at estimating the biological variation of serum GFAP. METHODS: Apparently healthy subjects (n=33) had blood sampled for three consecutive days. On the second day, blood was also drawn every third hour from 9 AM to 9 PM. Serum GFAP was measured by Single Molecule Array (Simoa™). Components of biological variation were estimated in a linear mixed-effects model. RESULTS: The overall median GFAP value was 92.5 pg/mL (range 34.4-260.3 pg/mL). The overall within- (CVI) and between-subject variations (CVG) were 9.7 and 39.5%. The reference change value was 36.9% for an increase. No day-to-day variation was observed, however semidiurnal variation was observed with increasing GFAP values between 9 AM and 12 PM (p<0.00001) and decreasing from 12 to 9 PM (p<0.001). CONCLUSIONS: Serum GFAP exhibits a relatively low CVI but a considerable CVG and a marked semidiurnal variation. This implies caution on the timing of blood sampling and when interpreting GFAP in relation to reference intervals, especially in conditions where only small GFAP differences are observed.

Biological variation of serum neurofilament light chain.

OBJECTIVES: The neurofilament light chain (NfL) has emerged as a versatile biomarker for CNS-diseases and is approaching clinical use. The observed changes in NfL levels are frequently of limited magnitude and in order to make clinical decisions based on NfL measurements, it is essential that biological variation is not confused with clinically relevant changes. The present study was designed to evaluate the biological variation of serum NfL. METHODS: Apparently healthy individuals (n=33) were submitted to blood draws for three days in a row. On the second day, blood draws were performed every third hour for 12 h. NfL was quantified in serum using the Simoa™ HD-1 platform. The within-subject variation (CVI) and between-subject variation (CVG) were calculated using linear mixed-effects models. RESULTS: The overall median value of NfL was 6.3 pg/mL (range 2.1-19.1). The CVI was 3.1% and the CVG was 35.6%. An increase in two serial measurements had to exceed 24.3% to be classified as significant at the 95% confidence level. Serum NfL levels remained stable during the day (p=0.40), whereas a minute variation (6.0-6.6 pg/mL) was observed from day-to-day (p=0.02). CONCLUSIONS: Serum NfL is subject to tight homeostatic regulation with none or neglectable semidiurnal and day-to-day variation, but considerable between-subject variation exists. This emphasizes serum NfL as a well-suited biomarker for disease monitoring, but warrants caution when interpreting NfL levels in relation to reference intervals in a diagnosis setting. Furthermore, NfL's tight regulation requires that the analytical variation is kept at a minimum.

Inflammatory Cytokines and ctDNA Are Biomarkers for Progression in Advanced-Stage Melanoma Patients Receiving Checkpoint Inhibitors.

PURPOSE: Checkpoint inhibitors have significantly improved treatment of metastatic melanoma. However, 40-60% of patients do not respond to therapy, emphasizing the need for better predictive biomarkers for treatment response to immune checkpoint inhibitors. Prorammed death-ligand 1(PD-L1) expression in tumor cells is currently used as a predictive biomarker; however, it lacks specificity. Therefore, it is of utmost importance to identify other novel biomarkers that can predict treatment outcome. EXPERIMENTAL DESIGN: We studied a small cohort of 16 patients with advanced-stage melanoma treated with first-line checkpoint inhibitors. Plasma samples were collected prior to treatment initiation and continuously during the first year of treatment. Circulating tumor DNA (ctDNA) level and the expression of ten inflammatory cytokines were analyzed. RESULTS: We found that the ctDNA-level in a blood sample collected after 6-8 weeks of therapy is predictive for response to checkpoint inhibitors. Patients with undetectable ctDNA had significantly longer progression-free survival (PFS) compared with patients with detectable ctDNA (median 26.3 vs. 2.1 months, p = 0.006). In parallel, we identified that high levels of the cytokines monocyte chemoattractant protein 1 (MCP1) and tumor necrosis factor a(TNFa) in baseline blood samples were significantly associated with longer PFS compared to low level of these cytokines (median not reached vs. 8.2 months p = 0.0008). CONCLUSIONS: These findings suggest that the levels of ctDNA, MCP1, and TNFa in baseline and early follow-up samples can predict disease progression in metastatic melanoma patients treated with checkpoint inhibitors. Potentially, these minimally invasive biomarkers may identify responders from non-responders.

Genomic Profiling of Circulating Tumor DNA Predicts Outcome and Demonstrates Tumor Evolution in ALK-Positive Non-Small Cell Lung Cancer Patients.

With the rapid development of targeted therapies for the treatment of cancer, methods for predicting response and outcome are in high demand. Non-small cell lung cancer driven by genomic rearrangements of the anaplastic lymphoma kinase (ALK) gene can be successfully treated with ALK-targeted therapy. Unfortunately, a subset of patients does not respond, and all patients ultimately acquire resistance, highlighting the need for better clinical tools to manage these patients. Here, we performed targeted next-generation sequencing on plasma circulating tumor DNA (ctDNA) from 24 patients to assess the clinical utility of ctDNA genomic profiling. Patients with detectable ctDNA prior to treatment had worse progression-free survival (PFS) than those without (median 8.7 vs. 15.2 months, p = 0.028). In addition, the presence of ctDNA within two months after treatment initiation predicted inferior PFS (median 4.6 vs. 14.5 months, p = 0.028). Longitudinal monitoring of ctDNA with droplet digital PCR during treatment reflected the radiological response and revealed potential acquired resistance mutations. Interestingly, an increase in the ctDNA concentration was evident prior to the determination of progressive disease by conventional radiological imaging, with a median lead time of 69 days (range 30-113). Genomic profiling of ctDNA is a promising tool for predicting outcome and monitoring response to targeted therapy.

Day-to-day and within-day biological variation of cell-free DNA.

BACKGROUND: Numerous studies have shown that cell-free DNA (cfDNA) levels may serve as a non-invasive biomarker of a broad spectrum of acute and chronic pathologies. However, in order to make clinical decisions based on cfDNA measurements, it is essential to understand the magnitude of biological variation so this variation is not confused with a variation that actually represent a clinically relevant change. The present study was designed to evaluate the biological variation of cfDNA in healthy subjects and lung cancer patients. METHODS: Plasma samples were collected from 33 healthy subjects and ten lung cancer patients over three days, as well as during the same day. CfDNA was quantified using droplet digital PCR. Biological variation data was estimated using mixed models. FINDINGS: The within-subject variation was 25% and the between-subject variation was 30%. The reference change value for the healthy subjects was 70%. There was no systematic difference in cfDNA levels from day-to-day (p = 0⋅61), but there was a significant decline during the day (p<0⋅01). The within-subject variation in cancer patients was comparable to healthy subjects, whereas the between-subject variation was much larger (139%). No systematic differences from day-to-day were observed for the cancer patients (p>0⋅3). INTERPRETATION: Our findings show that cfDNA levels fluctuate significantly during the day and exhibit considerable within-subject variation. Thus, the data presented offer a substantial contribution to the interpretation of the clinical significance of cfDNA. FUNDING: Læge Sofus Carl Emil Friis og hustru Olga Doris Friis' Legat, Harboefonden, and Dagmar Marshalls Fond.

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.

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.

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.

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.

IGF1R depletion facilitates MET-amplification as mechanism of acquired resistance to erlotinib in HCC827 NSCLC cells.

EGFR-mutated non-small cell lung cancer patients experience relapse within 1-2 years of treatment with EGFR-inhibitors, such as erlotinib. Multiple resistance mechanisms have been identified including secondary EGFR-mutations, MET-amplification, and epithelial-mesenchymal transition (EMT). Previous studies have indicated a role of Insulin-like growth factor 1 receptor (IGF1R) in acquired resistance to EGFR-directed drugs as well as in EMT. In the present study, we have investigated the involvement of IGF1R in acquired high-dose erlotinib resistance in the EGFR-mutated lung adenocarcinoma cell line HCC827. We observed that IGF1R was upregulated in the immediate response to erlotinib and hyperactivated in erlotinib resistant HCC827 cells. Resistant cells additionally acquired features of EMT, whereas MET-amplification and secondary EGFR-mutations were absent. Using CRISPR/Cas9, we generated a HCC827(IGFR1-/-) cell line and subsequently investigated resistance development in response to high-dose erlotinib. Interestingly, HCC827(IGFR1-/-) cells were now observed to specifically amplify the MET gene. Additionally, we observed a reduced level of mesenchymal markers in HCC827(IGFR1-/-) indicating an intrinsic enhanced epithelial signature compared to HCC827 cells. In conclusion, our data show that IGF1R have an important role in defining selected resistance mechanisms in response to high doses of erlotinib.