Epithelial-to-mesenchymal transition (EMT) is a key process in cancer progression and metastasis, requiring cooperation of the epidermal growth factor/Ras with the transforming growth factor-β ...(TGF-β) signaling pathway in a multistep process. The molecular mechanisms by which Ras signaling contributes to EMT, however, remain elusive to a large extent. We therefore examined the transcriptional repressor Ets2-repressor factor (ERF)-a bona fide Ras-extracellular signal-regulated kinase/mitogen-activated protein kinase effector-for its ability to interfere with TGF-β-induced EMT in mammary epithelial cells (EpH4) expressing oncogenic Ras (EpRas). ERF-overexpressing EpRas cells failed to undergo TGF-β-induced EMT, formed three-dimensional tubular structures in collagen gels, and retained expression of epithelial markers. Transcriptome analysis indicated that TGF-β signaling through Smads was mostly unaffected, and ERF suppressed the TGF-β-induced EMT via Semaphorin-7a repression. Forced expression of Semaphorin-7a in ERF-overexpressing EpRas cells reestablished their ability to undergo EMT. In contrast, inhibition of Semaphorin-7a in the parental EpRas cells inhibited their ability to undergo TGF-β-induced EMT. Our data suggest that oncogenic Ras may play an additional role in EMT via the ERF, regulating Semaphorin-7a and providing a new interconnection between the Ras- and the TGF-β-signaling pathways.
Metastatic melanoma is a deadly skin cancer and is resistant to almost all existing treatment. Vemurafenib, which targets the BRAFV600E mutation, is one of the drugs that improves patient outcome, ...but the patients next develop secondary resistance and a return to cancer. Thus, new therapeutic strategies are needed to treat melanomas and to increase the duration of v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) inhibitor response. The ERK pathway controls cell proliferation, and Aurora B plays a pivotal role in cell division. Here, we confirm that Aurora B is highly expressed in metastatic melanoma cells and that Aurora B inhibition triggers both senescence-like phenotypes and cell death in melanoma cells. Furthermore, we show that the BRAF/ERK axis controls Aurora B expression at the transcriptional level, likely through the transcription factor FOXM1. Our results provide insight into the mechanism of Aurora B regulation and the first molecular basis of Aurora B regulation in melanoma cells. The inhibition of Aurora B expression that we observed in vemurafenib-sensitive melanoma cells was rescued in cells resistant to this drug. Consistently, these latter cells remain sensitive to the effect of the Aurora B inhibitor. Noteworthy, wild-type BRAF melanoma cells are also sensitive to Aurora B inhibition. Collectively, our findings, showing that Aurora B is a potential target in melanoma cells, particularly in those vemurafenib-resistant, may open new avenues to improve the treatment of metastatic melanoma.
Background: BRAFV600E melanoma cells develop resistance to vemurafenib. The BRAF/ERK axis controls melanoma cell proliferation. Aurora B is a key actor of mitosis.
Results: The BRAF/ERK axis regulates Aurora B. Vemurafenib-resistant melanoma cells are sensitive to Aurora B inhibition.
Conclusion: Aurora B is a valuable target in melanoma cells.
Significance: Our findings provide insights into Aurora B regulation and on new druggable targets to overcome vemurafenib resistance.
As new SARS-CoV-2 variants emerge, there is an urgent need to increase the efficiency and availability of viral genome sequencing, notably to detect the lineage in samples with a low viral load. ...SARS-CoV-2 genome next-generation sequencing (NGS) was performed retrospectively in a single center on 175 positive samples from individuals. An automated workflow used the Ion AmpliSeq SARS-CoV-2 Insight Research Assay on the Genexus Sequencer. All samples were collected in the metropolitan area of the city of Nice (France) over a period of 32 weeks (from 19 July 2021 to 11 February 2022). In total, 76% of cases were identified with a low viral load (Ct ≥ 32, and ≤200 copies/µL). The NGS analysis was successful in 91% of cases, among which 57% of cases harbored the Delta variant, and 34% the Omicron BA.1.1 variant. Only 9% of cases had unreadable sequences. There was no significant difference in the viral load in patients infected with the Omicron variant compared to the Delta variant (Ct values,
= 0.0507; copy number,
= 0.252). We show that the NGS analysis of the SARS-CoV-2 genome provides reliable detection of the Delta and Omicron SARS-CoV-2 variants in low viral load samples.
The introduction of liquid biopsies for the detection of
mutations in non-small cell lung cancer patients (NSCLC) has revolutionized the clinical care. However, liquid biopsies are technically ...challenging and require specifically trained personnel. To facilitate the implementation of liquid biopsies for the detection of
mutations from plasma, we have assessed a fully automated cartridge-based qPCR test that allows the automatic detection of
mutations directly from plasma. We have analyzed 54 NSCLC patients and compared the results of the cartridge-base device to an FDA-approved assay. Detection of EGFR mutations was comparable but slightly lower in the cartridge-based device for L858R mutations (14/15 detected, 93%) and exon 19 deletions (18/20 detected, 90%). Unfortunately, 8/54 (15%) tests failed but increasing the proteinase K volume helped to recover 3/4 (75%) unsuccessful samples. In summary, the fully automated cartridge-based device allowed the detection of EGFR mutations directly from plasma in NSCLC patients with promising accuracy. However, protocol adjustments are necessary to reduce a high test failure rate.
NGS from plasma samples in non-squamous cell lung carcinoma (NSCC) can aid in the detection of actionable genomic alterations. However, the absolute clinical value of NGS in liquid biopsy (LB) made ...at baseline is currently uncertain. We assessed the impact of plasma-based NGS using an in-house test and an outsourced test in comparison to a routine molecular pathology workflow.
Twenty-four advanced/metastatic treatment-naïve NSCC patients were prospectively included. NGS analyses were conducted both in-house using the Oncomine cfTNA Panel and in an external testing center using the Foundation Liquid assay. NGS analysis and/or specific molecular based assays were conducted in parallel on tissue or cytological samples.
Both LB tests were well correlated. Tissue NGS results were obtained in 67% of patients and demonstrated good correlation with LB assays. Activating EGFR mutations were detected using LB tests in three patients. PD-L1 expression assessed in tissue sections enabled the initiation of pembrolizumab treatment in five patients.
NGS from LB is feasible in routine clinical practice using an in-house or an outsourced test at baseline. However, the impact on therapy selection was limited in this small series of patients and LB was not able to replace tissue-based testing in our hands.
Ophthalmic malignancies include various rare neoplasms involving the conjunctiva, the uvea, or the periocular area. These tumors are characterized by their scarcity as well as their histological, and ...sometimes genetic, diversity. Uveal melanoma (UM) is the most common primary intraocular malignancy. UM raises three main challenges highlighting the specificity of ophthalmic malignancies. First, UM is a very rare malignancy with an estimated incidence of 6 cases per million inhabitants. Second, tissue biopsy is not routinely recommended due to the risk of extraocular dissemination. Third, UM is an aggressive cancer because it is estimated that about 50% of patients will experience metastatic spread without any curative treatment available at this stage. These challenges better explain the two main objectives in the creation of a dedicated UM biobank. First, collecting UM samples is essential due to tissue scarcity. Second, large-scale translational research programs based on stored human samples will help to better determine UM pathogenesis with the aim of identifying new biomarkers, allowing for early diagnosis and new targeted treatment modalities. Other periocular malignancies, such as conjunctival melanomas or orbital malignancies, also raise specific concerns. In this context, the number of biobanks worldwide dedicated to ocular malignancies is very limited. The aims of this article were (i) to describe the specific challenges raised by a dedicated ocular malignancy biobank, (ii) to report our experience in setting up such a biobank, and (iii) to discuss future perspectives in this field.
The number of genomic alterations required for targeted therapy of non-squamous non-small cell lung cancer (NS-NSCLC) patients has increased and become more complex these last few years. These ...molecular abnormalities lead to treatment that provides improvement in overall survival for certain patients. However, these treated tumors inexorably develop mechanisms of resistance, some of which can be targeted with new therapies. The characterization of the genomic alterations needs to be performed in a short turnaround time (TAT), as indicated by the international guidelines. The origin of the tissue biopsies used for the analyses is diverse, but their size is progressively decreasing due to the development of less invasive methods. In this respect, the pathologists are facing a number of different challenges requiring them to set up efficient molecular technologies while maintaining a strategy that allows rapid diagnosis. We report here our experience concerning the development of an optimal workflow for genomic alteration assessment as reflex testing in routine clinical practice at diagnosis for NS-NSCLC patients by using an ultra-fast-next generation sequencing approach (Ion Torrent Genexus Sequencer, Thermo Fisher Scientific). We show that the molecular targets currently available to personalized medicine in thoracic oncology can be identified using this system in an appropriate TAT, notably when only a small amount of nucleic acids is available. We discuss the new challenges and the perspectives of using such an ultra-fast NGS in daily practice.
The introduction of liquid biopsy using PCR-based assays into routine practice has had a strong impact on the treatment of EGFR-mutated lung adenocarcinoma and is now commonly used for routine ...testing of EGFR mutations in certain clinical settings. To assess whether the claimed benefits of PCR-based assays hold true in daily practice at a multicenter clinical institution, we assessed how treatment decisions are affected by PCR-based assays for the analysis of EGFR mutations from plasma samples in a centralized laboratory (LPCE, Nice, France).
A total of 345 samples were analyzed using the US Food and Drug Administration–approved Cobas EGFR Mutation Test v2 and 103 using the Therascreen EGFR Plasma RGQ PCR Kit over 3 years (395 samples from 324 patients). Eleven plasma samples were validated independently using Cobas at 3 institutions, and 130 samples were analyzed using Stilla digital PCR. Clinical data were collected for 175 (54%) of 324 patients.
Cobas was superior to the Therascreen assay and demonstrated 100% reproducibility. Digital PCR showed only 48%, 83%, and 58% concordance with Cobas for exon 19 deletions, L858R mutations, and T790M mutations, respectively. Liquid biopsies helped inform and change treatment when resistance occurred and enabled the detection of EGFR mutations in patients when biopsy tissue results were unavailable.
PCR-based assays are a fast and convenient test, allowing the detection of primary and secondary EGFR mutations from plasma. Cobas proved to be a reliable test, whereas digital PCR produced too many inconclusive results to be currently recommended as a principal testing device.
We assessed EGFR mutations using liquid biopsies (LB) in 345 patients with lung cancer to analyze if this approach is reliable and sufficiently robust to guide rapid treatment decisions in daily practice. LB for the assessment of EGFR mutations can guide treatment decisions, but some limitations must be carefully communicated to the physicians to avoid unnecessary testing.
The current diagnostic standard for coronavirus disease 2019 (COVID-19) is reverse transcriptase-polymerase chain reaction (RT-PCR) testing with nasopharyngeal (NP) swabs. The invasiveness and need ...for trained personnel make the NP technique unsuited for repeated community-based mass screening. We developed a technique to collect saliva in a simple and easy way with the sponges that are usually used for tamponade of epistaxis. This study was carried out to validate the clinical performance of oral sponge (OS) sampling for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing.
Over a period of 22 weeks, we collected prospectively 409 paired NP and OS samples from consecutive subjects presenting to a public community-based free screening centre. Subjects were referred by their attending physician because of recent COVID-19 symptoms (n = 147) or by the contact tracing staff of the French public health insurance because they were considered as close contacts of a laboratory-confirmed COVID-19 case (n = 262).
In symptomatic subjects, RT-PCR SARS-CoV-2 testing with OS showed a 96.5% (95% CI: 89.6-94.8) concordance with NP testing, and a 93.2% (95% CI: 89.1-97.3) sensitivity when using the IdyllaTM platform and a sensitivity of 76.3% (95% CI: 69.4-83.2) on the Synlab Barla laboratory platform. In close contacts the NP-OS concordance (93.8%, 95% CI: 90.9-96.7) and OS sensitivity (71.9%, 95% CI: 66.5-77.3) were slightly lower.
These results strongly suggest that OS testing is a straightforward, low-cost and high-throughput sampling method that can be used for frequent RT-PCR testing of COVID-19 patients and mass screening of populations.
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