Differential Formation of Stress Granules in Radiosensitive and Radioresistant Head and Neck Squamous Cell Carcinoma Cells.

PURPOSE: Stress granules (SGs) are cytoplasmic aggregates in which mRNAs and specific proteins are trapped in response to a variety of damaging agents. They participate in the cellular defense mechanisms. Currently, their mechanism of formation in response to ionizing radiation and their role in tumor-cell radiosensitivity remain elusive. METHODS AND MATERIALS: The kinetics of SG formation was investigated after the delivery of photon irradiation at different doses to head and neck squamous cell carcinoma cell lines with different radiosensitivities and the HeLa cervical cancer cell line (used as reference). In parallel, the response to a canonical inducer of SGs, sodium arsenite, was also studied. Immunolabeling of SG-specific proteins and mRNA fluorescence in situ hybridization enabled SG detection and quantification. Furthermore, a ribopuromycylation assay was used to assess the cell translational status. To determine whether reactive oxygen species were involved in SG formation, their scavenging or production was induced by pharmacologic pretreatment in both SCC61 and SQ20B cells. RESULTS: Photon irradiation at different doses led to the formation of cytoplasmic foci that were positive for different SG markers. The presence of SGs gradually increased from 30 minutes to 2 hours postexposure in HeLa, SCC61, and Cal60 radiosensitive cells. In turn, the SQ20B and FaDu radioresistant cells did not form SGs. These results indicated a correlation between sensitivity to photon irradiation and SG formation. Moreover, SG formation was significantly reduced by reactive oxygen species scavenging using dimethyl sulfoxide in SCC61 cells, which supported their role in SG formation. However, a reciprocal experiment in SQ20B cells that depleted glutathione using buthionine sulfoximide did not restore SG formation in these cells. CONCLUSIONS: SGs are formed in response to irradiation in radiosensitive, but not in radioresistant, head and neck squamous cell carcinoma cells. Interestingly, compared with sodium arsenite-induced SGs, photon-induced SGs exhibited a different morphology and cellular localization. Moreover, photon-induced SGs were not associated with the inhibition of translation; rather, they depended on oxidative stress.

Circulating H3K27 Methylated Nucleosome Plasma Concentration: Synergistic Information with Circulating Tumor DNA Molecular Profiling.

The molecular profiling of circulating tumor DNA (ctDNA) is a helpful tool not only in cancer treatment, but also in the early detection of relapse. However, the clinical interpretation of a ctDNA negative result remains challenging. The characterization of circulating nucleosomes (carrying cell-free DNA) and associated epigenetic modifications (playing a key role in the tumorigenesis of different cancers) may provide useful information for patient management, by supporting the contributive value of ctDNA molecular profiling. Significantly elevated concentrations of H3K27Me3 nucleosomes were found in plasmas at the diagnosis, and during the follow-up, of NSCLC patients, compared to healthy donors (p-value < 0.0001). By combining the H3K27Me3 level and the ctDNA molecular profile, we found that 25.5% of the patients had H3K27Me3 levels above the cut off, and no somatic alteration was detected at diagnosis. This strongly supports the presence of non-mutated ctDNA in the corresponding plasma. During the patient follow-up, a high H3K27Me3-nucleosome level was found in 15.1% of the sample, despite no somatic mutations being detected, allowing the identification of disease progression from 43.1% to 58.2% over molecular profiling alone. Measuring H3K27Me3-nucleosome levels in combination with ctDNA molecular profiling may improve confidence in the negative molecular result for cfDNA in lung cancer at diagnosis, and may also be a promising biomarker for molecular residual disease (MRD) monitoring, during and/or after treatment.

Paired Comparison of Routine Molecular Screening of Patient Samples with Advanced Non-Small Cell Lung Cancer in Circulating Cell-Free DNA Using Three Targeted Assays.

INTRODUCTION: Progressive advanced non-small cell lung cancer (NSCLC) accounts for about 80-85% of all lung cancers. Approximately 10-50% of patients with NSCLC harbor targetable activating mutations, such as in-frame deletions in Exon 19 (Ex19del) of EGFR. Currently, for patients with advanced NSCLC, testing for sensitizing mutations in EGFR is mandatory prior to the administration of tyrosine kinase inhibitors. PATIENTS AND METHODS: Plasma was collected from patients with NSCLC. We carried out targeted NGS using the Plasma-SeqSensei™ SOLID CANCER IVD kit on cfDNA (circulating free DNA). Clinical concordance for plasma detection of known oncogenic drivers was reported. In a subset of cases, validation was carried out using an orthogonal OncoBEAMTM EGFR V2 assay, as well as with our custom validated NGS assay. Somatic alterations were filtered, removing somatic mutations attributable to clonal hematopoiesis for our custom validated NGS assay. RESULTS: In the plasma samples, driver targetable mutations were studied, with a mutant allele frequency (MAF) ranging from 0.00% (negative detection) to 82.25%, using the targeted next-generation sequencing Plasma-SeqSensei™ SOLID CANCER IVD Kit. In comparison with the OncoBEAMTM EGFR V2 kit, the EGFR concordance is 89.16% (based on the common genomic regions). The sensitivity and specificity rates based on the genomic regions (EGFR exons 18, 19, 20, and 21) were 84.62% and 94.67%. Furthermore, the observed clinical genomic discordances were present in 25% of the samples: 5% in those linked to the lower of coverage of the OncoBEAMTM EGFR V2 kit, 7% in those induced by the sensitivity limit on the EGFR with the Plasma-SeqSensei™ SOLID CANCER IVD Kit, and 13% in the samples linked to the larger KRAS, PIK3CA, BRAF coverage of the Plasma-SeqSensei™ SOLID CANCER IVD kit. Most of these somatic alterations were cross validated in our orthogonal custom validated NGS assay, used in the routine management of patients. The concordance is 82.19% in the common genomic regions (EGFR exons 18, 19, 20, 21; KRAS exons 2, 3, 4; BRAF exons 11, 15; and PIK3CA exons 10, 21). The sensitivity and specificity rates were 89.38% and 76.12%, respectively. The 32% of genomic discordances were composed of 5% caused by the limit of coverage of the Plasma-SeqSensei™ SOLID CANCER IVD kit, 11% induced by the sensitivity limit of our custom validated NGS assay, and 16% linked to the additional oncodriver analysis, which is only covered by our custom validated NGS assay. CONCLUSIONS: The Plasma-SeqSensei™ SOLID CANCER IVD kit resulted in de novo detection of targetable oncogenic drivers and resistance alterations, with a high sensitivity and accuracy for low and high cfDNA inputs. Thus, this assay is a sensitive, robust, and accurate test.

The 'stealth-bomber' paradigm for deciphering the tumour response to carbon-ion irradiation.

Numerous studies have demonstrated the higher biological efficacy of carbon-ion irradiation (C-ions) and their ballistic precision compared with photons. At the nanometre scale, the reactive oxygen species (ROS) produced by radiation and responsible for the indirect effects are differentially distributed according to the type of radiation. Photon irradiation induces a homogeneous ROS distribution, whereas ROS remain condensed in clusters in the C-ions tracks. Based on this linear energy transfer-dependent differential nanometric ROS distribution, we propose that the higher biological efficacy and specificities of the molecular response to C-ions rely on a 'stealth-bomber' effect. When biological targets are on the trajectories of the particles, the clustered radicals in the tracks are responsible for a 'bomber' effect. Furthermore, the low proportion of ROS outside the tracks is not able to trigger the cellular mechanisms of defence and proliferation. The ability of C-ions to deceive the cellular defence of the cancer cells is then categorised as a 'stealth' effect. This review aims to classify the biological arguments supporting the paradigm of the 'stealth-bomber' as responsible for the biological superiority of C-ions compared with photons. It also explains how and why C-ions will always be more efficient for treating patients with radioresistant cancers than conventional radiotherapy.

Circulating Tumor Cell Detection during Neoadjuvant Chemotherapy to Predict Early Response in Locally Advanced Oropharyngeal Cancers: A Prospective Pilot Study.

Patients with locally advanced oropharyngeal carcinoma treated with neoadjuvant chemotherapy are reassessed both radiologically and clinically to adapt their treatment after the first cycle. However, some responders show early tumor progression after adjuvant radiotherapy. This cohort study evaluated circulating tumor cells (CTCs) from a population of locally advanced oropharyngeal carcinoma patients treated with docetaxel, cisplatin, and 5-fluorouracil (DCF) induction chemotherapy or DCF with a modified dose and fractioned administration. The counts and phenotypes of CTCs were assessed at baseline and at day 21 of treatment, after isolation using the RosetteSepTM technique based on negative enrichment. At baseline, 6 out of 21 patients had CTCs (28.6%). On day 21, 5 out of 11 patients had CTCs (41.6%). There was no significant difference in the overall and progression-free survival between patients with or without CTCs at baseline (p = 0.44 and 0.78) or day 21 (p = 0.88 and 0.5). Out of the 11 patients tested at day 21, 4 had a positive variation of CTCs (33%). Patients with a positive variation of CTCs display a lower overall survival. Our findings suggest that the variation in the number of CTCs would be a better guide to the management of treatment, with possible early changes in treatment strategy.

CD44, γ-H2AX, and p-ATM Expressions in Short-Term Ex Vivo Culture of Tumour Slices Predict the Treatment Response in Patients with Oral Squamous Cell Carcinoma.

Squamous cell carcinoma is the most common type of head and neck cancer (HNSCC) with a disease-free survival at 3 years that does not exceed 30%. Biomarkers able to predict clinical outcomes are clearly needed. The purpose of this study was to investigate whether a short-term culture of tumour fragments irradiated ex vivo could anticipate patient responses to chemo- and/or radiotherapies. Biopsies were collected prior to treatment from a cohort of 28 patients with non-operable tumours of the oral cavity or oropharynx, and then cultured ex vivo. Short-term biopsy slice culture is a robust method that keeps cells viable for 7 days. Different biomarkers involved in the stemness status (CD44) or the DNA damage response (pATM and γ-H2AX) were investigated for their potential to predict the treatment response. A higher expression of all these markers was predictive of a poor response to treatment. This allowed the stratification of responder or non-responder patients to treatment. Moreover, the ratio for the expression of the three markers 24 h after 4 Gy irradiation versus 0 Gy was higher in responder than in non-responder patients. Finally, combining these biomarkers greatly improved their predictive potential, especially when the γ-H2AX ratio was associated with the CD44 ratio or the pATM ratio. These results encourage further evaluation of these biomarkers in a larger cohort of patients.

A Review of the Role of Hypoxia in Radioresistance in Cancer Therapy.

Hypoxia involves neoplastic cells. Unlike normal tissue, solid tumors are composed of aberrant vasculature, leading to a hypoxic microenvironment. Hypoxia is also known to be involved in both metastasis initiation and therapy resistance. Radiotherapy is the appropriate treatment in about half of all cancers, but loco-regional control failure and a disease recurrence often occur due to clinical radioresistance. Hypoxia induces radioresistance through a number of molecular pathways, and numerous strategies have been developed to overcome this. Nevertheless, these strategies have resulted in disappointing results, including adverse effects and limited efficacy. Additional clinical studies are needed to achieve a better understanding of the complex hypoxia pathways. This review presents an update on the mechanisms of hypoxia in radioresistance in solid tumors and the potential therapeutic solutions.

Involvement of HIF-1α in the Detection, Signaling, and Repair of DNA Double-Strand Breaks after Photon and Carbon-Ion Irradiation.

Hypoxia-Inducible Factor 1α (HIF-1α), which promotes cancer cell survival, is the main regulator of oxygen homeostasis. Hypoxia combined with photon and carbon ion irradiation (C-ions) stabilizes HIF-1α. Silencing HIF-1α under hypoxia leads to substantial radiosensitization of Head-and-Neck Squamous Cell Carcinoma (HNSCC) cells after both photons and C-ions. Thus, this study aimed to clarify a potential involvement of HIF-1α in the detection, signaling, and repair of DNA Double-Strand-Breaks (DSBs) in response to both irradiations, in two HNSCC cell lines and their subpopulations of Cancer-Stem Cells (CSCs). After confirming the nucleoshuttling of HIF-1α in response to both exposure under hypoxia, we showed that silencing HIF-1α in non-CSCs and CSCs decreased the initiation of the DSB detection (P-ATM), and increased the residual phosphorylated H2AX (γH2AX) foci. While HIF-1α silencing did not modulate 53BP1 expression, P-DNA-PKcs (NHEJ-c) and RAD51 (HR) signals decreased. Altogether, our experiments demonstrate the involvement of HIF-1α in the detection and signaling of DSBs, but also in the main repair pathways (NHEJ-c and HR), without favoring one of them. Combining HIF-1α silencing with both types of radiation could therefore present a potential therapeutic benefit of targeting CSCs mostly present in tumor hypoxic niches.

Combining radiation to EGFR and Bcl-2 blockade: a new approach to target cancer stem cells in head and neck squamous cell carcinoma.

PURPOSE: The clinical outcome of head and neck squamous cell carcinoma (HNSCC) remains poor, partly due to the presence of resistant cancer stem cells (CSCs) which are responsible of recurrences. CSCs have low EGFR expression and, conversely, overexpress the anti-apoptotic Bcl-2 protein, which is involved in resistance to apoptosis and the invasion/migration capacities of tumour cells. METHODS: The combination therapy of ABT-199, a Bcl-2 inhibitor, cetuximab an EGFR inhibitor, and radiation using an HNSCC model (SQ20B cell line) and its corresponding CSC subpopulation were evaluated in vitro (2D/3D cell proliferation; invasion/migration and apoptosis using videomicroscopy) and in vivo. RESULTS: Cetuximab strongly inhibited 2D and 3D cell proliferation, as well as invasion/migration, only in non-CSC-SQ20B cells, whereas ABT-199 selectively inhibited these mechanisms in SQ20B/CSCs. The combination of irradiation + cetuximab + ABT-199 increased the inhibition of the 2D and 3D cell proliferation, invasion/migration, and resistance to apoptosis in both cell sub-populations. In addition, in a nude mouse model with heterotopic tumour xenograft, a treatment combining cetuximab + ABT-199 with fractional irradiation strongly delayed the tumour growth and increased in vivo lifespan without side effects. CONCLUSION: Based on the present results, this triple combination therapy may represent a new opportunity for testing in clinical trials, particularly in locally advanced HNSCC.

Impact of hypoxia on the double-strand break repair after photon and carbon ion irradiation of radioresistant HNSCC cells.

DNA double-strand breaks (DSBs) induced by photon irradiation are the most deleterious damage for cancer cells and their efficient repair may contribute to radioresistance, particularly in hypoxic conditions. Carbon ions (C-ions) act independently of the oxygen concentration and trigger complex- and clustered-DSBs difficult to repair. Understanding the interrelation between hypoxia, radiation-type, and DNA-repair is therefore essential for overcoming radioresistance. The DSBs signaling and the contribution of the canonical non-homologous end-joining (NHEJ-c) and homologous-recombination (HR) repair pathways were assessed by immunostaining in two cancer-stem-cell (CSCs) and non-CSCs HNSCC cell lines. Detection and signaling of DSBs were lower in response to C-ions than photons. Hypoxia increased the decay-rate of the detected DSBs (γH2AX) in CSCs after photons and the initiation of DSB repair signaling (P-ATM) in CSCs and non-CSCs after both radiations, but not the choice of DSB repair pathway (53BP1). Additionally, hypoxia increased the NHEJ-c (DNA-PK) and the HR pathway (RAD51) activation only after photons. Furthermore, the involvement of the HR seemed to be higher in CSCs after photons and in non-CSCs after C-ions. Taken together, our results show that C-ions may overcome the radioresistance of HNSCC associated with DNA repair, particularly in CSCs, and independently of a hypoxic microenvironment.

Development of a new expanded next-generation sequencing panel for genetic diseases involved in dyslipidemia.

The aim of this study was to provide an efficient tool: reliable, able to increase the molecular diagnosis performance, to facilitate the detection of copy number variants (CNV), to assess genetic risk scores (wGRS) and to offer the opportunity to explore candidate genes. Custom SeqCap EZ libraries, NextSeq500 sequencing and a homemade pipeline enable the analysis of 311 dyslipidemia-related genes. In the training group (48 DNA from patients with a well-established molecular diagnosis), this next-generation sequencing (NGS) workflow showed an analytical sensitivity >99% (n = 532 variants) without any false negative including a partial deletion of one exon. In the prospective group, from 25 DNA from patients without prior molecular analyses, 18 rare variants were identified in the first intention panel genes, allowing the diagnosis of monogenic dyslipidemia in 11 patients. In six other patients, the analysis of minor genes and wGRS determination provided a hypothesis to explain the dyslipidemia. Remaining data from the whole NGS workflow identified four patients with potentially deleterious variants. This NGS process gives a major opportunity to accede to an enhanced understanding of the genetic of dyslipidemia by simultaneous assessment of multiple genetic determinants.

Involvement of a homozygous exon 6 deletion of LMF1 gene in intermittent severe hypertriglyceridemia.

Severe hypertriglyceridemia (HTG), characterized by triglycerides (TG) permanently over 10 mmol/L, may correspond to familial chylomicronemia syndrome (FCS), a rare disorder. However, hypertriglyceridemic patients more often present multifactorial chylomicronemia syndrome (MCS), characterized by highly variable TG. A few nonsense variants of LMF1 gene were reported in literature in FCS patients. In this study, we described a woman with an intermittent severe HTG. NGS analysis and the sequencing of a long range PCR product revealed a homozygous deletion of 6507 base pairs in LMF1 gene, c.730-1528_898-3417del, removing exon 6, predicted to create an in-frame deletion of 56 amino acids, p.(Thr244_Gln299del). Despite an exon 6 homozygous deletion of LMF1, the patient's highly variable lipid phenotype was suggestive of MCS diagnosis.

Radiation-induced bystander and abscopal effects: important lessons from preclinical models.

Radiotherapy is a pivotal component in the curative treatment of patients with localised cancer and isolated metastasis, as well as being used as a palliative strategy for patients with disseminated disease. The clinical efficacy of radiotherapy has traditionally been attributed to the local effects of ionising radiation, which induces cell death by directly and indirectly inducing DNA damage, but substantial work has uncovered an unexpected and dual relationship between tumour irradiation and the host immune system. In clinical practice, it is, therefore, tempting to tailor immunotherapies with radiotherapy in order to synergise innate and adaptive immunity against cancer cells, as well as to bypass immune tolerance and exhaustion, with the aim of facilitating tumour regression. However, our understanding of how radiation impacts on immune system activation is still in its early stages, and concerns and challenges regarding therapeutic applications still need to be overcome. With the increasing use of immunotherapy and its common combination with ionising radiation, this review briefly delineates current knowledge about the non-targeted effects of radiotherapy, and aims to provide insights, at the preclinical level, into the mechanisms that are involved with the potential to yield clinically relevant combinatorial approaches of radiotherapy and immunotherapy.

Dual inhibition of DNA-PK and DNA polymerase theta overcomes radiation resistance induced by p53 deficiency.

TP53 deficiency in cancer is associated with poor patient outcomes and resistance to DNA damaging therapies. However, the mechanisms underlying treatment resistance in p53-deficient cells remain poorly characterized. Using live cell imaging of DNA double-strand breaks (DSBs) and cell cycle state transitions, we show that p53-deficient cells exhibit accelerated repair of radiomimetic-induced DSBs arising in S phase. Low-dose DNA-dependent protein kinase (DNA-PK) inhibition increases the S-phase DSB burden in p53-deficient cells, resulting in elevated rates of mitotic catastrophe. However, a subset of p53-deficient cells exhibits intrinsic resistance to radiomimetic-induced DSBs despite DNA-PK inhibition. We show that p53-deficient cells under DNA-PK inhibition utilize DNA polymerase theta (Pol θ)-mediated end joining repair to promote their viability in response to therapy-induced DSBs. Pol θ inhibition selectively increases S-phase DSB burden after radiomimetic therapy and promotes prolonged G2 arrest. Dual inhibition of DNA-PK and Pol θ restores radiation sensitivity in p53-deficient cells as well as in p53-mutant breast cancer cell lines. Thus, combination targeting of DNA-PK- and Pol θ-dependent end joining repair represents a promising strategy for overcoming resistance to DNA damaging therapies in p53-deficient cancers.

Profiling of circulating tumor DNA in plasma of non-small cell lung cancer patients, monitoring of epidermal growth factor receptor p.T790M mutated allelic fraction using beads, emulsion, amplification, and magnetics companion assay and evaluation in future application in mimicking circulating tumor cells.

Cell-free plasma DNA (cfDNA) and mimicking circulating tumor cells (mCTCs) have demonstrated tremendous potential for molecular diagnosis of cancer and have been rapidly implemented in specific settings. However, widespread clinical adoption still faces some obstacles. The purpose was to compare the performance of a BEAMing (beads, emulsion, amplification, and magnetics) assay (OncoBEAM™-epidermal growth factor receptor [EGFR] [Sysmex Inostics]) and a next-generation sequencing assay (NGS; 56G Oncology panel kit, Swift Bioscience) to detect the p.T790M EGFR mutation in cfDNA of non-small cell lung cancer (NSCLC) patients. CfDNA samples (n = 183) were collected within our hospital from patients having a known EGFR sensitizing mutation, and presenting disease progression while under first-line therapy. EGFR mutations were detected using NGS in 42.1% of samples during progression in cfDNA. Testing using the OncoBEAM™-EGFR assay enabled detection of the p.T790M EGFR mutation in 40/183 NSCLC patients (21.8%) versus 20/183 (10.9%), using the NGS assay. Samples that were only positive with the OncoBEAM™-EGFR assay had lower mutant allelic fractions (Mean = 0.1304%; SD ± 0.1463%). In addition, we investigated the detection of p.T790M in mCTCs using H1975 cells. These cells spiked into whole blood were enriched using the ClearCellFX1 microfluidic device. Using the OncoBEAM™-EGFR assay, p.T790M was detected in as few as 1.33 tumoral cells/mL. Overall, these findings highlight the value of using the OncoBEAM™-EGFR to optimize detection of the p.T790M mutation, as well as the complementary clinical value that each of the mutation detection assay offers: NGS enabled the detection of mutations in other oncogenes that may be relevant to secondary resistance mechanisms, whereas the OncoBEAM™-EGFR assay achieved higher sensitivity for detection of clinically actionable mutations.

ROS Production and Distribution: A New Paradigm to Explain the Differential Effects of X-ray and Carbon Ion Irradiation on Cancer Stem Cell Migration and Invasion.

Although conventional radiotherapy promotes the migration/invasion of cancer stem cells (CSCs) under normoxia, carbon ion (C-ion) irradiation actually decreases these processes. Unraveling the mechanisms of this discrepancy, particularly under the hypoxic conditions that pertain in niches where CSCs are preferentially localized, would provide a better understanding of the origins of metastases. Invasion/migration, proteins involved in epithelial-to-mesenchymal transition (EMT), and expression of MMP-2 and HIF-1α were quantified in the CSC subpopulations of two head-and-neck squamous cell carcinoma (HNSCC) cell lines irradiated with X-rays or C-ions. X-rays triggered HNSCC-CSC migration/invasion under normoxia, however this effect was significantly attenuated under hypoxia. C-ions induced fewer of these processes in both oxygenation conditions. The differential response to C-ions was associated with a lack of HIF-1α stabilization, MMP-2 expression, or activation of kinases of the main EMT signaling pathways. Furthermore,we demonstrated a major role of reactive oxygen species (ROS) in the triggering of invasion/migration in response to X-rays. Monte-Carlo simulations demonstrated that HO● radicals are quantitatively higher after C-ions than after X-rays, however they are very differently distributed within cells. We postulate that the uniform distribution of ROS after X-rays induces the mechanisms leading to invasion/migration, which ROS concentrated in C-ion tracks are unable to trigger.

Cross-platform comparison for the detection of RAS mutations in cfDNA (ddPCR Biorad detection assay, BEAMing assay, and NGS strategy).

CfDNA samples from colon (mCRC) and non-small cell lung cancers (NSCLC) (CIRCAN cohort) were compared using three platforms: droplet digital PCR (ddPCR, Biorad); BEAMing/OncoBEAM™-RAS-CRC (Sysmex Inostics); next-generation sequencing (NGS, Illumina), utilizing the 56G oncology panel (Swift Biosciences). Tissue biopsy and time matched cfDNA samples were collected at diagnosis in the mCRC cohort and during 1st progression in the NSCLC cohort. Excellent matches between cfDNA/FFPE mutation profiles were observed. Detection thresholds were between 0.5-1% for cfDNA samples examined using ddPCR and NGS, and 0.03% with BEAMing. This high level of sensitivity enabled the detection of KRAS mutations in 5/19 CRC patients with negative FFPE profiles. In the mCRC cohort, comparison of mutation results obtained by testing FFPE to those obtained by testing cfDNA by ddPCR resulted in 47% sensitivity, 77% specificity, 70% positive predictive value (PPV) and 55% negative predictive value (NPV). For BEAMing, we observed 93% sensitivity, 69% specificity, 78% PPV and 90% NPV. Finally, sensitivity of NGS was 73%, specificity was 77%, PPV 79% and NPV 71%. Our study highlights the complementarity of different diagnostic approaches and variability of results between OncoBEAM™-RAS-CRC and NGS assays. While the NGS assay provided a larger breadth of coverage of the major targetable alterations of 56 genes in one run, its performance for specific alterations was frequently confirmed by ddPCR results.

[Dihydropyrimidine déhydrogenase (DPD) deficiency screening and securing of fluoropyrimidine-based chemotherapies: Update and recommendations of the French GPCO-Unicancer and RNPGx networks]. / Dépistage du déficit en dihydropyrimidine déshydrogénase (DPD) et sécurisation des chimiothérapies à base de fluoropyrimidines : mise au point et recommandations nationales du GPCO-Unicancer et du RNPGx.

Fluoropyrimidines (FU) are still the most prescribed anticancer drugs for the treatment of solid cancers. However, fluoropyrimidines cause severe toxicities in 10 to 40% of patients and toxic deaths in 0.2 to 0.8% of patients, resulting in a real public health problem. The main origin of FU-related toxicities is a deficiency of dihydropyrimidine dehydrogenase (DPD), the rate-limiting enzyme of 5-FU catabolism. DPD deficiency may be identified through pharmacogenetics testing including phenotyping (direct or indirect measurement of enzyme activity) or genotyping (detection of inactivating polymorphisms on the DPYD gene). Approximately 3 to 15% of patients exhibit a partial deficiency and 0.1 to 0.5% a complete DPD deficiency. Currently, there is no regulatory obligation for DPD deficiency screening in patients scheduled to receive a fluoropyrimidine-based chemotherapy. Based on the levels of evidence from the literature data and considering current French practices, the Group of Clinical Pharmacology in Oncology (GPCO)-UNICANCER and the French Network of Pharmacogenetics (RNPGx) recommend the following: (1) to screen DPD deficiency before initiating any chemotherapy containing 5-FU or capecitabine; (2) to perform DPD phenotyping by measuring plasma uracil (U) concentrations (possibly associated with dihydrouracil/U ratio), and DPYD genotyping (variants *2A, *13, p.D949V, HapB3); (3) to reduce the initial FU dose (first cycle) according to DPD status, if needed, and further, to consider increasing the dose at subsequent cycles according to treatment tolerance. In France, 17 public laboratories currently undertake routine screening of DPD deficiency.

Evaluation of the Cell Invasion and Migration Process: A Comparison of the Video Microscope-based Scratch Wound Assay and the Boyden Chamber Assay.

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration and invasion abilities to understand how cancer cells disseminate, with the aim of developing new treatment strategies. Analysis of the cellular and molecular basis of these abilities has led to the characterization of cell mobility and the physicochemical properties of the cytoskeleton and cellular microenvironment. For many years, the Boyden chamber assay and the scratch wound assay have been the standard techniques to study cell invasion and migration. However, these two techniques have limitations. The Boyden chamber assay is difficult and time consuming, and the scratch wound assay has low reproducibility. Development of modern technologies, especially in microscopy, has increased the reproducibility of the scratch wound assay. Using powerful analysis systems, an "in-incubator" video microscope can be used to provide automatic and real-time analysis of cell migration and invasion. The aim of this paper is to report and compare the two assays used to study cell invasion and migration: the Boyden chamber assay and an optimized in vitro video microscope-based scratch wound assay.

Evaluation of pre-analytical conditions and comparison of the performance of several digital PCR assays for the detection of major EGFR mutations in circulating DNA from non-small cell lung cancers: the CIRCAN_0 study.

Non invasive somatic detection assays are suitable for repetitive tumor characterization or for detecting the appearance of somatic resistance during lung cancer. Molecular diagnosis based on circulating free DNA (cfDNA) offers the opportunity to track the genomic evolution of the tumor, and was chosen to assess the molecular profile of several EGFR alterations, including deletions in exon 19 (delEX19), the L858R substitution on exon 21 and the EGFR resistance mutation T790M on exon 20. Our study aimed at determining optimal pre-analytical conditions and EGFR mutation detection assays for analyzing cfDNA using the picoliter-droplet digital polymerase chain reaction (ddPCR) assay. Within the framework of the CIRCAN project set-up at the Lyon University Hospital, plasma samples were collected to establish a pre-analytical and analytical workflow of cfDNA analysis. We evaluated all of the steps from blood sampling to mutation detection output, including shipping conditions (4H versus 24H in EDTA tubes), the reproducibility of cfDNA extraction, the specificity/sensitivity of ddPCR (using external controls), and the comparison of different PCR assays for the detection of the three most important EGFR hotspots, which highlighted the increased sensitivity of our in-house primers/probes. Hence, we have described a new protocol facilitating the molecular detection of somatic mutations in cancer patients from liquid biopsies, improving their diagnosis and introducing a less traumatic monitoring system during tumor progression.