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The origin of metastases is a topic that has sparked controversy. Despite recent advancements, metastatic disease continues to pose challenges. The first admitted model of how metastases develop revolves around cells breaking away from the primary tumor, known as circulating tumor cells (CTCs). These cells survive while circulating through the bloodstream and subsequently establish themselves in secondary organs, a process often referred to as the "metastatic cascade". This intricate and dynamic process involves various steps, but all the mechanisms behind metastatic dissemination are not yet comprehensively elucidated. The "seed and soil" theory has shed light on the phenomenon of metastatic organotropism and the existence of pre-metastatic niches. It is now established that these niches can be primed by factors secreted by the primary tumor before the arrival of CTCs. In particular, exosomes have been identified as important contributors to this priming. Another concept then emerged, i.e. the "genometastasis" theory, which challenged all other postulates. It emphasizes the intriguing but promising role of cell-free DNA (cfDNA) in metastasis formation through oncogenic formation of recipient cells. However, it cannot be ruled out that all these theories are intertwined. This review outlines the primary theories regarding the metastases formation that involve CTCs, and depicts cfDNA, a potential second player in the metastasis formation. We discuss the potential interrelationships between CTCs and cfDNA, and propose both in vitro and in vivo experimental strategies to explore all plausible theories. [ABSTRACT FROM AUTHOR]

Pancreatic cancer is one of the most aggressive diseases with a very poor outcome. Olaparib, a PARP inhibitor, as maintenance therapy showed benefits in patients with metastatic pancreatic adenocarcinoma bearing germline BRCA1/2 mutations. However, germline BRCA mutation has been described in only 4–7% of patients with pancreatic adenocarcinoma. A CRISPR/Cas9-mediated system was used to knock-in the c.763G > T p.(Glu255*) and c.2133C > A p.(Cys711*) mutations in cell lines to obtain truncated BRCA1 and BRCA2 proteins, respectively. A CRISPR/Cas9 ribonucleoprotein complex was assembled for each mutation and transfected into two pancreatic cell lines (T3M4 and Capan-2) and into a breast cancer cell lines (MCF7) as control. BRCA protein levels were significantly decreased in all BRCA-depleted cells (P < 0.05), proving the transfection efficiency of our CRISPR/Cas9 systems. As expected, the calculated olaparib IC50 were significantly reduced for all cell lines harbored BRCA1 or BRCA2 mutations compared to wild-type BRCA1/2 cells (P < 0.01). Furthermore, we observed a higher induction of apoptosis after 72 h olaparib treatment in BRCA-depleted cells than in wild-type cells. This strategy might offer new insights into the management of patients with pancreatic cancer and open up new perspectives based on the in vivo use of CRISPR/Cas9 strategy. [ABSTRACT FROM AUTHOR]

Gene fusions and MET exon skipping drive oncogenesis in 8–9% and 3% of non-small cell lung cancers (NSCLC) respectively. Their detection are essential for the management of patients since they confer sensitivity to specific targeted therapies with significant clinical benefit over conventional chemotherapy. Immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) account for historical reference techniques however molecular-based technologies (RNA-based sequencing and RT-PCR) are emerging as alternative or complementary methods. Here, we evaluated the analytical performance of the fully-automated RT-PCR Idylla GeneFusion assay compared to reference methods using 35 fixed NSCLC samples. Idylla demonstrated overall agreement, sensitivity and specificity of 100% compared to RNASeq. Interestingly, it succeeded in retrieving 10 out of 11 samples with inconclusive results due to insufficient RNA quality for sequencing. Idylla showed an overall agreement, sensitivity and specificity of 90.32%, 91.67% and 89.47% compared to IHC/FISH respectively. Using commercial standards, the limit of detection of the Idylla system for the most frequent fusions and exon skipping ranges between 5 and 10 ng RNA input. These results support that the Idylla assay is a reliable and rapid option for the detection of these alterations, however a particular attention is needed for the interpretation of the expression imbalance. [ABSTRACT FROM AUTHOR]

The assessment of EGFR mutations is recommended for the management of patients with non-small cell lung cancer (NSCLC). Presence of EGFR mutation is associated with response or resistance to EGFR tyrosine kinase inhibitors (EGFR-TKI). Liquid biopsy is nowadays widely used for the detection of resistance to EGFR-TKI. We evaluated here the performance of the Idylla ctEGFR mutation assay for the detection of EGFR mutations in circulating tumour DNA (ctDNA) in plasma from patients with NSCLC. Previously characterized plasma samples from 38 patients with NSCLC were analysed using 2 different analytical conditions (C1 and C2). The limit of detection (LOD) was evaluated using 2 mL of healthy donor plasma spiked with commercial DNA controls. Overall agreement, sensitivity and specificity were 92.1%, 86.7% and 95.7% for C1 condition respectively and 94.7%, 86.7% and 100% for C2 condition respectively. The T790M secondary resistance mutation was detected in two samples out of 3. The Idylla system was able to detect the exon 19 deletion from 6 copies/mL and up to 91 copies/mL for the G719S mutation. These results support that the Idylla ctEGFR mutation assay is a rapid option for the detection of EGFR hotspots mutations in plasma samples, however a particular attention is needed for its interpretation. [ABSTRACT FROM AUTHOR]

Radiation-induced fibrosis (RIF) is the main late radiation toxicity in breast cancer patients. Most of the current 3D in vitro breast cancer models are composed by cancer cells only and are unable to reproduce the complex cellular homeostasis within the tumor microenvironment to study RIF mechanisms. In order to account complex cellular interactions within the tumor microenvironment, an advanced 3D spheroid model, consisting of the luminal breast cancer MCF-7 cells and MRC-5 fibroblasts, was developed. The spheroids were generated using the liquid overlay technique in culture media into 96-well plates previously coated with 1% agarose (m/v, in water). In total, 21 experimental setups were tested during the optimization of the model. The generated spheroids were characterized using fluorescence imaging, immunohistology and immunohistochemistry. The expression of ECM components was confirmed in co-culture spheroids. Using α-SMA staining, we confirmed the differentiation of healthy fibroblasts into myofibroblasts upon the co-culturing with cancer cells. The induction of fibrosis was studied in spheroids treated 24 h with 10 ng/mL TGF-β and/or 2 Gy irradiation. Overall, the developed advanced 3D stroma-rich in vitro model of breast cancer provides a possibility to study fibrosis mechanisms taking into account 3D arrangement of the complex tumor microenvironment. [ABSTRACT FROM AUTHOR]

Background: Metastatic or unresectable melanoma is a serious and deadly disease. Anti-BRAF and immunotherapy improved overall survival in patients with metastatic disease. Thus, BRAF genotyping is important to choose the right therapy. Methods: In our study, we assessed and compared BRAF mutations in 59 formalin-fixed and paraffin-embedded tumor samples of patients with metastatic melanoma with next-generation sequencing (NGS), Cobas 4800 BRAF V600 mutation test CE-IVD commercial kit, high-resolution melting PCR (HRM), multiplex real-time allele specific amplification (multiplexed RT-ASA) and immunohistochemistry (IHC). Results: Thirty-one samples were found bearing a BRAF mutation with NGS (52.5%), 28 with Cobas test (47.5%), 28 with HRM (47.5%), 29 with multiplexed RT-ASA (49.2%) and 27 with IHC (45.8%). Based on NGS data, 26 (81.2%) were c.1799 T>A (p.Val600Glu), 3 (9.4%) were c. 1798-1799 GT>AA (p.Val600Lys), 1 was c.1789_1790 CT>TC (p.Leu597Ser) and 2 were complex mutations. Sensitivity was 90.3% for Cobas test, 93.1% for multiplexed RT-ASA and 87.1% for IHC and HRM. Specificity was 100% for Cobas test, IHC and multiplexed RT-ASA and 96.4% for HRM. The reference assay was NGS. Rare mutations were detected with NGS and HRM: c.1789_1790 CT>TC (p.Leu597Ser) mutation and the complex mutation c.1796 A>T; c.1797_1798 insACT (p.Thr599Thr; p.Thr599_Val600insThr). Our data suggest that multiplexed RT-ASA is the most sensitive assay but specific primers for each mutation are needed. HRM can detect all exon 15 mutations but has a lower sensitivity. Because of its specificity for Val600Glu mutation, IHC may be considered only as a screening tool and testing should be completed by a method able to detect other V600 mutations. BRAF Cobas assay is Val600Glu-specific and has poor sensitivity for the other V600 mutations; thus, it looks important to use multiplex assays able to detect all V600 mutations because a false-negative result will deprive the patient of an important treatment option. [ABSTRACT FROM AUTHOR]

Background: Overall survival of metastatic colorectal cancer (mCRC) patients has been improved with the addition of targeted therapy such as anti-epithelial growth factor receptor monoclonal antibodies (anti-EGFR mAbs) to standard chemotherapy. Retrospective studies and randomized trials showed that the presence of RAS mutations was linked to the absence of clinical response to anti-EGFR mAbs. Patients harboring KRAS and NRAS mutations on exons 2, 3 or 4 have little or no benefit from anti-EGFR therapies. Polymerase chain reaction (PCR)-based assays are routinely used to assess KRAS and NRAS status, whereas deep sequencing with next generation sequencing (NGS) currently represents an alternative method.Objective: The objective of our study was to identify KRAS and NRAS non-hotspot mutations using NGS of mCRC tumor samples.Method: DNA was extracted from 188 consecutive formalin-fixed paraffin embedded samples of histologically proven colorectal cancer tumor tissue from patients with mCRC. Following amplification, DNA was sequenced by ultra-deep pyrosequencing. Non-hotspot mutations identified by NGS (frequency of mutated allele range [1.8-70.6 %]) were confirmed by Sanger direct-sequencing when possible.Results: NGS procedure was applicable in 94 % of the cases and detected mutations in 62 % of the samples. Nine uncommon mutational profiles were found with a frequency of mutated allele  > 1 %. Silent mutations were found in 3.6 % of the samples. Mutations at or near functional domains of RAS proteins, other than defined hotspots, were found in 3.6 %. NGS proved to be accurate, sensitive and suitable for routine RAS genotyping.Conclusion: Clinical responses to anti-EGFR mAbs are potentially impaired in the presence of these uncommon RAS mutations. [ABSTRACT FROM AUTHOR]

Many studies documented the influence of KRAS mutation status on the response of patients with metastatic colorectal cancer (mCRC) to anti-EGFR monoclonal antibodies. The COBAS 4800 KRAS is an assay using real time PCR and TaqMelt technology, CE-IVD validated, for the detection of 19 KRAS somatic mutations in exons 2 and 3. We compared COBAS with previously validated PCR TaqMan and High Resolution Melting (HRM) assays on 156 formalin-fixed paraffin embedded (FFPE) specimens of colorectal carcinoma. DNA extraction procedures, using the Qiagen QiAMP kit and the Roche COBAS DNA kit, were also compared. Of the 156 samples, 132 were interpretable using COBAS and TaqMan and 92 using COBAS and HRM. No statistically significant difference was found between COBAS/TaqMan and COBAS/HRM ( k = 0.937; p < 0.001 - four discordant cases were found, mostly concerning codon 61 mutations and k = 0.891; p < 0.001 - five discordant cases were found, three regarding codon 61 and two on codon 12/13, respectively). No difference was found between the two DNA extraction methods ( t = 1.7185; dol = 39; α = 5 %). The three assays were found suitable to detect accurately KRAS mutations in colon FFPE specimens. COBAS and TaqMan were found to be more robust than HRM, as they yielded fewer non-interpretable results. DNA extraction kits were found to provide equivalent results. The present study shows that pre-screening using COBAS with further TaqMan mutation characterization constitutes an easy and reliable approach for routine diagnostic purposes [ABSTRACT FROM AUTHOR]

New active drugs are needed for the treatment of primary brain tumors in both children and adults. S16020 is a cytotoxic olivacine derivative that inhibits topoisomerase II. The aim of the study was to determine its antitumor activity in athymic mice bearing subcutaneous medulloblastoma (IGRM33, 34, 57) and glioblastoma (IGRG88, 93, 121) xenografts treated at an advanced stage of tumor growth in comparison with that of doxorubicin. Animals were randomly assigned to receive i.v. S16020 or doxorubicin weekly for three consecutive weeks. The optimal dose was 80 mg/kg per week. S16020 demonstrated a significant antitumor activity in two out of three medulloblastoma xenografts. IGRM57 xenografts were highly sensitive with 100% tumor regressions and a tumor growth delay (TGD) of 102 days, while one of eight IGRM34 xenografts showed a partial regression with a TGD of 16 days. Doxorubicin was significantly more active than S16020 in these two models. IGRM33, a model established from a tumor in relapse after chemotherapy and radiotherapy, was refractory to both drugs. S16020 demonstrated a significant antitumor activity in the three glioblastoma xenografts evaluated. The wild-type p53 IGRG93 xenograft was highly sensitive with 100% tumor regressions and a TGD of 54 days. IGRG121 (wt p53) and IGRG88 (mutant p53) were moderately sensitive with TGDs of 33 and 23 days, respectively. Doxorubicin showed greater activity in two of these models. All six xenografts exhibited low expression of mdr1 as quantitated by RT-PCR, and no correlation was found with the activity of either drug. Conversely, a low activity of the two drugs was significantly associated with a high expression of MRP1 in medulloblastomas. Finally, no relationship was observed between drug sensitivity to either drug and expression of their target, topoisomerase IIα. In conclusion, S16020 and doxorubicin showed significant antitumor activity in brain tumor xenografts treated at an advanced stage of tumor growth. Their activity was related to MRP1 expression in medulloblastomas. [ABSTRACT FROM AUTHOR]

Polyethylenimine (PEI) derivatives are polycationic nonviral vectors for gene transfer. Previous results achieved in vitro in head and neck cancer cells demonstrated that glucosylated PEI yields higher gene transfer efficiency and longer transgene expression than unsubstituted PEI. Using glucosylated PEI, p53 gene transfer was successfully achieved with subsequent recovery of P53 protein expression and induction of spontaneous apoptosis. The present study reports in vivo data achieved in human head and neck squamous cell carcinoma xenografted mice. Using biotinylated PEI and histochemistry analysis, the vector was found to diffuse in the proliferating cells of the tumor tissue, sparing necrotic areas. No diffusion was observed inside keratinized area composed of nonproliferating, mature differentiated cells. Using green fluorescent protein (GFP) transfection and fluorescence microscopy, the transgene expression was mainly observed at the periphery of the tumor containing proliferating cells. GFP expression appeared lower inside the tumor depth. Quantitative transgene expression kinetics was then determined using luciferase as reporter gene. The maximal transgene expression was achieved 48 hours after intratumoral injection of glucosylated PEI/DNA complexes. The highest gene transfer efficacy was achieved 48 hours after two intratumoral injection. After transfection of wild-type p53, tumor growth inhibition was observed in tumor-bearing mice receiving intratumoral injection of glucosylated PEI/DNA complexes repeated twice weekly. Tumor growth inhibition was maintained under continuous treatment using the same schedule. In all experiments, no noticeable toxicity was observed. The present results demonstrate the feasibility and the tumor growth inhibition potency of nonviral gene transfer using glucosylated polyethylenimine. Cancer Gene Therapy (2002) 9, 708–714 doi:10.1038/sj.cgt.7700485 [ABSTRACT FROM AUTHOR]

Polyethylenimine (PEI) derivatives are potent polycationic nonviral vectors for gene transfer. The gene transfer efficiency of glucosylated and galactosylated PEI derivatives was optimized using green fluorescent protein gene as reporter gene in FaDu and PANC3 human carcinoma cell lines. Glucosylated or galactosylated PEI derivatives were found to be slightly less cytotoxic than unsubstituted PEI. Gene transfer efficiency was found to be related to DNA/cell number ratio and optimal gene transfer efficiency was achieved at 4 μg DNA/105 cells. PEI–DNA complexes were found to enter cells rapidly and were detected into cytoplasmic vesicles 2 hours post-transfection. Green fluorescent protein gene expression was detected 4–6 hours after transfection and reached maximal value 24 hours post-transfection. The results achieved demonstrated that glucosylated PEI yield higher and longer gene transfer efficiency than unsubstituted PEI. Using glucosylated PEI allowed to achieve significant gene transfer in more than 10% of the total cell population for more than 4 days. These data were then applied to p53 gene transfer in PANC3 cells bearing p53 gene deletion and consequently unable to initiate apoptosis. Using glucosylated PEI, p53 gene transfer was successfully achieved with subsequent recovery of p53 mRNA expression and transient P53 protein expression. P53 protein functionality was further demonstrated because transfected cells underwent apoptosis. Cancer Gene Therapy (2001) 8, 203–210 [ABSTRACT FROM AUTHOR]

Microsatellite instability (MSI) status is routinely assessed in patients with colorectal and endometrial cancers as it contributes to Lynch syndrome initial screening, tumour prognosis and selecting patients for immunotherapy. Currently, standard reference methods recommended for MSI/dMMR (deficient MisMatch Repair) testing consist of immunohistochemistry and pentaplex PCR-based assays, however, novel molecular-based techniques are emerging. Here, we aimed to evaluate the performance of a custom capture-based NGS method and the Bio-Rad ddPCR and Idylla approaches for the determination of MSI status for theranostic purposes in 30 formalin-fixed paraffin embedded (FFPE) tissue samples from patients with endometrial (n = 15) and colorectal (n = 15) cancers. All samples were previously characterised using IHC and Promega MSI Analysis System and these assays set as golden standard. Overall agreement, sensitivity and specificity of our custom-built NGS panel were 93.30%, 93.75% and 92.86% respectively. Overall agreement, sensitivity and specificity were 100% with the Idylla MSI system. The Bio-Rad ddPCR MSI assay showed a 100% concordance, sensitivity and specificity. The custom capture-based NGS, Bio-Rad ddPCR and Idylla approaches represent viable and complementary options to IHC and Promega MSI Analysis System for the detection of MSI. Bio-Rad ddPCR and Idylla MSI assays accounts for easy and fast screening assays while the NGS approach offers the advantages to simultaneously detect MSI and clinically relevant genomic alterations. [ABSTRACT FROM AUTHOR]

Diagnosis of lung cancer can sometimes be challenging and is of major interest since effective molecular-guided therapies are available. Compounds of tobacco smoke may generate a specific substitutional signature in lung, which is the most exposed organ. To predict whether a tumor is of lung origin or not, we developed and validated the EASILUNG (Exome And SIgnature LUNG) test based on the relative frequencies of somatic substitutions on coding non-transcribed DNA strands from whole-exome sequenced tumors. Data from 7,796 frozen tumor samples (prior to any treatment) from 32 TCGA solid cancer groups were used for its development. External validation was carried out on a local dataset of 196 consecutive routine exome results. Eight out of the 12 classes of substitutions were required to compute the EASILUNG signature that demonstrated good calibration and good discriminative power with a sensitivity of 83% and a specificity of 72% after recalibration on the external validation dataset. This innovative test may be helpful in medical decision-making in patients with unknown primary tumors potentially of lung origin and in the diagnosis of lung cancer in smokers. [ABSTRACT FROM AUTHOR]

RAS genotyping is mandatory to predict anti-EGFR monoclonal antibodies (mAbs) therapy resistance and BRAF genotyping is a relevant prognosis marker in patients with metastatic colorectal cancer. Although the role of hotspot mutations is well defined, the impact of uncommon mutations is still unknown. In this study, we aimed to discuss the potential utility of detecting uncommon RAS and BRAF mutation profiles with next-generation sequencing. A total of 779 FFPE samples from patients with metastatic colorectal cancer with valid NGS results were screened and 22 uncommon mutational profiles of KRAS, NRAS and BRAF genes were selected. In silico prediction of mutation impact was then assessed by 2 predictive scores and a structural protein modelling. Three samples carry a single KRAS non-hotspot mutation, one a single NRAS non-hotspot mutation, four a single BRAF non-hotspot mutation and fourteen carry several mutations. This in silico study shows that some non-hotspot RAS mutations seem to behave like hotspot mutations and warrant further examination to assess whether they should confer a resistance to anti-EGFR mAbs therapy for patients bearing these non-hotspot RAS mutations. For BRAF gene, non-V600E mutations may characterise a novel subtype of mCRC with better prognosis, potentially implying a modification of therapeutic strategy. [ABSTRACT FROM AUTHOR]