Mutational Characteristics of Primary Mucosal Melanoma: A Systematic Review.

BACKGROUND: Primary mucosal melanomas (PMMs) are rare and clinically heterogeneous, including head and neck (HNMs), vulvovaginal (VVMs), conjunctival (CjMs), anorectal (ARMs) and penile (PMs) melanomas. While the prognosis of advanced cutaneous melanoma has noticeably improved using treatments with immune checkpoint inhibitors (ICIs) and molecules targeting BRAF and MEK, few advances have been made for PMMs because of their poorer response to ICIs and their different genetic profile. This prompted us to conduct a systematic review of molecular studies of PMMs to clarify their pathogenesis and potential therapeutic targets. METHODS: All articles that examined gene mutations in PMMs were identified from the databases and selected based on predefined inclusion criteria. Mutation rate was calculated for all PMMs and each location group by relating the number of mutations identified to the total number of samples analysed. RESULTS: Among 1,581 studies identified, 88 were selected. Overall, the frequency of KIT, BRAF and NRAS mutation was 13.5%, 12.9% and 12.1%, respectively. KIT mutation ranged from 6.4% for CjMs to 16.6% for ARMs, BRAF mutation from 8.6% for ARMs to 31.1% for CjMs, and NRAS mutation from 6.2% for ARMs to 18.5% for CjMs. Among 101 other genes analysed, 33 had mutation rates over 10%, including TTN, TSC1, POM121, NF1, MTOR and SF3B1. CONCLUSION: In addition to BRAF, NRAS and KIT genes commonly studied, our systematic review identified significantly mutated genes that have already been associated (e.g., TSC1, mTOR, POLE or ATRX) or could be associated with (future) targeted therapies. PROSPERO ID: CRD42020185552.

Clinical assessment of the miR-34, miR-200, ZEB1 and SNAIL EMT regulation hub underlines the differential prognostic value of EMT miRs to drive mesenchymal transition and prognosis in resected NSCLC.

BACKGROUND: Patients with non-small cell lung cancer (NSCLC) receiving curative surgery have a risk of relapse, and adjuvant treatments only translate into a 5% increase in 5-year survival. We assessed the clinical significance of epithelial-mesenchymal transition (EMT) and explored its association with the [SNAIL/miR-34]:[ZEB/miR-200] regulation hub to refine prognostic information. METHODS: We validated a 7-gene EMT score using a consecutive series of 176 resected NSCLC. We quantified EMT transcription factors, microRNAs (miRs) of the miR-200, miR-34 families and miR-200 promoter hypermethylation to identify outcome predictors. RESULTS: Most tumours presented with an EMT-hybrid state and the EMT score was not predictive of outcome. Individually, all miR-200 were inversely associated with the EMT score, but only chromosome-1 miRs, miR-200a, b, 429, were associated with disease-free survival (p = 0.08, 0.05 and 0.025) and overall survival (p = 0.013, 0.003 and 0.006). We validated these associations on The Cancer Genome Atlas data. Tumour unsupervised clustering based on miR expression identified two good prognostic groups, unrelated to the EMT score, suggesting that miR profiling may have an important clinical value. CONCLUSION: miR-200 family members do not have similar predictive value. Core EMT-miR, regulators and not EMT itself, identify NSCLC patients with a low risk of relapse after surgery.

F4, a collagen XIX-derived peptide, inhibits tumor angiogenesis through αvß3 and α5ß1 integrin interaction.

We previously demonstrated that F4 peptide (CNPEDCLYPVSHAHQR) from collagen XIX was able to inhibit melanoma cell migrationin vitro and cancer progression in a mouse melanoma model. The aim of the present work was to study the anti-angiogenic properties of F4 peptide. We demonstrated that F4 peptide inhibited VEGF-induced pseudo-tube formation on Matrigel by endothelial cells and endothelial sprouting in a rat aortic ring assay. By affinity chromatography, we identified αvß3 and α5ß1 integrins as potential receptors for F4 peptide on endothelial cell surface. Using solid phase assays, we proved the direct interaction between F4 and both integrins. Taken together, our results demonstrate that F4 peptide is a potent antitumor agent inhibiting both angiogenesis and tumor cell migration.

Extracellular Vesicle-Dependent Cross-Talk in Cancer-Focus on Pancreatic Cancer.

Extracellular vesicles (EVs) like exosomes and shed microvesicles are generated by many different cells. However, among all the cells, cancer cells are now recognized to secrete more EVs than healthy cells. Tumor-derived EVs can be isolated from biofluids such as blood, urine, ascitic fluid, and saliva. Their numerous components (nucleic acids, proteins, and lipids) possess many pleiotropic functions involved in cancer progression. The tumor-derived EVs generated under the influence of tumor microenvironment play distant roles and promote cellular communication by directly interacting with different cells. Moreover, they modulate extracellular matrix remodeling and tumor progression. Tumor-derived EVs are involved in pre-metastatic niche formation, dependent on the EV-associated protein receptors, and in cancer chemoresistance as they transfer drug-resistance-related genes to recipient cells. Recent advances in preclinical and clinical fields suggest their potential use as biomarkers for diagnosis and prognosis as well as for drug delivery in cancer. In this Review, we discuss EV characteristics and pro-tumor capacities, and highlight the future crucial impact of tumor-derived EVs in pancreatic cancer diagnosis and prognosis.

Angiogenesis Inhibition by a Short 13 Amino Acid Peptide Sequence of Tetrastatin, the α4(IV) NC1 Domain of Collagen IV.

Angiogenesis is defined as the formation of new capillaries by sprouting from the pre-existing microvasculature. It occurs in physiological and pathological processes particularly in tumor growth and metastasis. α1, α2, α3, and α6 NC1 domains from type IV collagen were reported to inhibit tumor angiogenesis. We previously demonstrated that the α4 NC1 domain from type IV collagen, named Tetrastatin, inhibited tumor growth in a mouse melanoma model. The inhibitory activity was located in a 13 amino acid sequence named QS-13. In the present paper, we demonstrate that QS-13 decreases VEGF-induced-angiogenesis in vivo using the Matrigel plug model. Fluorescence molecular tomography allows the measurement of a 65% decrease in Matrigel plug angiogenesis following QS-13 administration. The results are confirmed by CD31 microvessel density analysis on Matrigel plug slices. QS-13 peptide decreases Human Umbilical Vein Endothelial Cells (HUVEC) migration and pseudotube formation in vitro. Relevant QS-13 conformations were obtained from molecular dynamics simulations and docking. A putative interaction of QS-13 with α5ß1 integrin was investigated. The interaction was confirmed by affinity chromatography, solid phase assay, and surface plasmon resonance. QS-13 binding site on α5ß1 integrin is located in close vicinity to the RGD binding site, as demonstrated by competition assays. Collectively, our results suggest that QS-13 exhibits a mighty anti-angiogenic activity that could be used in cancer treatment and other pathologies with excessive angiogenesis such as hemangioma, psoriasis or diabetes.

Immunotherapy in non-small-cell lung cancer: from targeted molecules to resistance patterns.

Immunotherapies are now considered as a pillar of non-small-cell lung cancer treatment. The main targets of immune-checkpoint inhibitors (ICI) are programmed cell death 1/programmed cell death ligand 1 and cytotoxic T-lymphocyte antigen 4, aiming at restoring antitumor immunity. Despite durable responses observed in some patients, all patients do not benefit from the treatment and almost all responders ultimately relapse after some time. In this review, we discuss the biomarkers that could be used to predict response to ICI, the current indications of ICI in non-small-cell lung cancer, the mechanisms inducing tumor-cell intrinsic or extrinsic resistance to ICI and finally, the potential treatment response monitoring.

Tumor Microenvironment: Extracellular Matrix Alterations Influence Tumor Progression.

The tumor microenvironment (TME) is composed of various cell types embedded in an altered extracellular matrix (ECM). ECM not only serves as a support for tumor cell but also regulates cell-cell or cell-matrix cross-talks. Alterations in ECM may be induced by hypoxia and acidosis, by oxygen free radicals generated by infiltrating inflammatory cells or by tumor- or stromal cell-secreted proteases. A poorer diagnosis for patients is often associated with ECM alterations. Tumor ECM proteome, also named cancer matrisome, is strongly altered, and different ECM protein signatures may be defined to serve as prognostic biomarkers. Collagen network reorganization facilitates tumor cell invasion. Proteoglycan expression and location are modified in the TME and affect cell invasion and metastatic dissemination. ECM macromolecule degradation by proteases may induce the release of angiogenic growth factors but also the release of proteoglycan-derived or ECM protein fragments, named matrikines or matricryptins. This review will focus on current knowledge and new insights in ECM alterations, degradation, and reticulation through cross-linking enzymes and on the role of ECM fragments in the control of cancer progression and their potential use as biomarkers in cancer diagnosis and prognosis.

Molecular markers and prediction of response to immunotherapy in non-small cell lung cancer, an update.

Immunotherapy represents one of the most promising therapeutic approaches in lung cancer, however 50% of lung cancer patients will not respond to this treatment, while others will have transitory or durable responses. Because side effects may be life threatening and treatment costs remain very high, the identification of predictive markers is mandatory and actually extensively studied. Factors that determine response to immune checkpoint inhibitors (ICI) are numerous including tumor microenvironment, immune tumor infiltrates, expression of immune checkpoint proteins (PD-1/PD-L1), gene expression signatures and molecular tumor profiles. Based on high impact factor publications and recent literature this review focuses on the potential predictive value of tumor molecular alterations and tumor mutation burden as predictive markers of response or resistance to ICI. We also discuss the role of circulating tumor DNA (ctDNA) to monitor ICI responses and propose an algorithm that integrates molecular markers upcoming recommendations for first line treatment.

Conformation-dependent binding of a Tetrastatin peptide to αvß3 integrin decreases melanoma progression through FAK/PI3K/Akt pathway inhibition.

Tetrastatin, a 230 amino acid sequence from collagen IV, was previously demonstrated to inhibit melanoma progression. In the present paper, we identified the minimal active sequence (QKISRCQVCVKYS: QS-13) that reproduced the anti-tumor effects of whole Tetrastatin in vivo and in vitro on melanoma cell proliferation, migration and invasion. We demonstrated that QS-13 binds to SK-MEL-28 melanoma cells through the αvß3 integrin using blocking antibody and ß3 integrin subunit siRNAs strategies. Relevant QS-13 conformations were extracted from molecular dynamics simulations and their interactions with αVß3 integrin were analyzed by docking experiments to determine the binding areas and the QS-13 amino acids crucial for the binding. The in silico results were confirmed by in vitro experiments. Indeed, QS-13 binding to SK-MEL-28 was dependent on the presence of a disulfide-bound as shown by mass spectroscopy and the binding site on αVß3 was located in close vicinity to the RGD binding site. QS-13 binding inhibits the FAK/PI3K/Akt pathway, a transduction pathway that is largely involved in tumor cell proliferation and migration. Taken together, our results demonstrate that the QS-13 peptide binds αvß3 integrin in a conformation-dependent manner and is a potent antitumor agent that could target cancer cells through αVß3.

Current and Future Molecular Testing in NSCLC, What Can We Expect from New Sequencing Technologies?

Recent changes in lung cancer care, including new approvals in first line and the introduction of high-throughput molecular technologies in routine testing led us to question ourselves on how deeper molecular testing may be helpful for the optimal use of targeted drugs. In this article, we review recent results in the scope of personalized medicine in lung cancer. We discuss biomarkers that have a therapeutic predictive value in lung cancer with a focus on recent changes and on the clinical value of large scale sequencing strategies. We review the use of second- and third-generation EGFR and ALK inhibitors with a focus on secondary resistance alterations. We discuss anti-BRAF and anti-MEK combo, emerging biomarkers as NRG1 and NTRKs fusions and immunotherapy. Finally, we discuss the different technical issues of comprehensive molecular profiling and show how large screenings might refine the prediction value of individual markers. Based on a review of recent publications (2012⁻2018), we address promising approaches for the treatment of patients with lung cancers and the technical challenges associated with the identification of new predictive markers.

Type XIX collagen: A new partner in the interactions between tumor cells and their microenvironment.

Type XIX collagen is a minor collagen that is associated with the basement membrane zone that belongs to the FACIT family (Fibril-Associated Collagens with Interrupted Triple helices). The FACIT family is composed of type IX, XII, XIV, XVI, XX, XXI, XXII and XIX collagens, which share many highly conserved structural motifs: a short NC1 domain, a thrombospondin-like N-terminal domain (TSPN), and numerous cysteine residues. The main role of FACITs is to ensure the integrity and stability of the extracellular matrix and its fibrillar collagen network by regulating the formation and size of the collagen fibrils. Type XIX collagen was discovered in a human rhabdomyosarcoma cell line. The collagen α1(XIX) chain is composed of 5 triple-helical domains (COL) interrupted by 6 non-triple-helical (NC) domains with a short, C-terminal, 19 amino acid non-collagenous domain (NC1). This collagen is involved in the differentiation of muscle cells, central nervous system development, and formation of the esophagus. Type XIX collagen is associated with the basement membrane zone, like type XVIII and XV collagens. Its short NC1(XIX) C-terminal domain inhibits the migration and invasion of melanoma cells. It also exerts a strong anti-angiogenic effect by inhibiting MMP-14 and VEGF expression. NC1(XIX) binding to αvß3 integrin decreases the phosphorylation of proteins involved in the FAK (Focal Adhesion Kinase)/PI3K (PhosphoInositide 3-Kinase)/Akt (protein kinase B)/mTOR (Mammalian Target Of Rapamycin) pathway. On the other hand, NC1(XIX) induces an increase in GSK3ß activity by decreasing its level of phosphorylation. The inhibition of this pathway could explain the anti-tumor properties of the NC1(XIX) domain.

Evaluation of Lumipulse® G1200 for the measurement of six tumor markers: Comparison with AIA® 2000.

Tumor marker assays are daily practiced, for screening and follow up of cancers. Interassay precision is an important parameter for the interpretation of the kinetics of the markers, in order to conclude to the efficiency or failure of treatment. The aim of this study was to compare two automated Immunoassay analyzers, Lumipulse® G1200 and AIA® 2000. Both analyzers used an immunoassay system but with different antibodies. Six tumor markers commonly used were studied: AFP, PSA, CA 19-9, CA 15-3, CA 125 and CEA. 253 samples have been collected over a period of one month and analyzed by both analyzers. Regression of Passing-Badblock and Bland-Altman diagram were used to analyze the results for AFP (n=36), PSA (n=39), CA-125 (n=40), CA 15-3 (n=40), CA 19-9 (n=46) and CEA (n=52) were performed. Analytical performances of Lumipulse® G1200 highlighted the good inter-run and intra-run precision of the analyzer. We obtained a good correlation coefficient between Lumipulse G1200® and AIA 2000®, >0.96 for most markers except CA 19-9 which provided a correlation coefficient significantly lower than that obtained with other markers. The concordance for all markers was >94% except for CA 19-9 (83.7%). This study showed a good correlation between the two analyzers and, therefore, a transfer from one analyzer to the other is possible for the different markers studied. However, we found here the classical difficulty to transfer this type of analysis, due to the absence of method standardization. This difficulty was particularly illustrated by CA19-9.

Analytical interference in the therapeutic drug monitoring of methotrexate.

High-dose of methotrexate chemotherapy is used in the treatment of some tumors. It presents several side effects that required therapeutic drug monitoring, which is commonly performed on 24, 48 and 72h after the beginning of the methotrexate infusion. Treatment of overexposure to methotrexate is based on injection of carboxypeptidase G2, which specifically degrades methotrexate into inactive metabolite: DAMPA. FPIA immunoassay on TDx automated analyzer (Abbott™) was used for therapeutic drug monitoring of methotrexate. This immunoassay presented a significant cross-reactivity between methotrexate and DAMPA, which widely overestimate the residual concentration compared to the gold standard HPLC/MS. TDx automated analyzer was substituted by a new immunoassay on Architect automated analyzer (Abbott™). However, this immunoassay has the same cross-reactivity, which needs to be careful when monitoring methotrexate after an injection of carboxypeptidase G2. In order to determine the most suitable assay for the therapeutic drug monitoring of methotrexate, the knowledge of injection of carboxypeptidase G2 remains essential.

The anti-tumor NC1 domain of collagen XIX inhibits the FAK/ PI3K/Akt/mTOR signaling pathway through αvß3 integrin interaction.

Type XIX collagen is a minor collagen associated with basement membranes. It was isolated for the first time in a human cDNA library from rhabdomyosarcoma and belongs to the FACITs family (Fibril Associated Collagens with Interrupted Triple Helices). Previously, we demonstrated that the NC1 domain of collagen XIX (NC1(XIX)) exerts anti-tumor properties on melanoma cells by inhibiting their migration and invasion. In the present work, we identified for the first time the integrin αvß3 as a receptor of NC1(XIX). Moreover, we demonstrated that NC1(XIX) inhibits the FAK/PI3K/Akt/mTOR pathway, by decreasing the phosphorylation and activity of the major proteins involved in this pathway. On the other hand, NC1(XIX) induced an increase of GSK3ß activity by decreasing its degree of phosphorylation. Treatments targeting this central signaling pathway in the development of melanoma are promising and new molecules should be developed. NC1(XIX) seems to have the potential for the design of new anti-cancer drugs.

[An acute monoclonal gammopathy?]. / Une gammapathie monoclonale d'apparition brutale ?

Serum protein electrophoresis is commonly used in case of acute or chronic renal failure. It can lead to the etiologic diagnosis by detecting monoclonal gammopathies which are frequently complicated by renal failure, such as cast nephropathy, Randall's disease or amyloidosis, or to explore an associated inflammatory syndrome. We report the occurrence of two monoclonal components in a patient without any monoclonal component 10 days earlier. The sudden appearance of these two monoclonal components associated to the context of sepsis of urinary origin suggested the diagnosis of transient monoclonal gammopathy. This hypothesis was confirmed by monitoring serum protein electrophoresis that showed a gradual decrease of these two monoclonal components few weeks after the resolution of the infectious disease. The main etiological factors of transient monoclonal gammopathies are infectious or autoimmune diseases. In this context, it is important to delay the achievement of serum protein electrophoresis after the acute episode, in order to avoid to falsely conclude to hematologic malignancy diagnosis. This can prevent costly biological examinations of these transient monoclonal gammopathies and invasive procedures like bone marrow examination.

Plasmin releases the anti-tumor peptide from the NC1 domain of collagen XIX.

During tumor invasion, tumor cells degrade the extracellular matrix. Basement membrane degradation is responsible for the production of peptides with anti-tumor properties. Type XIX collagen is associated with basement membranes in vascular, neuronal, mesenchymal and epithelial tissues. Previously, we demonstrated that the non-collagenous NC1, C-terminal, domain of collagen XIX [NC1(XIX)] inhibits the migration capacities of tumor cells and exerts a strong inhibition of tumor growth. Here, we demonstrate that plasmin, one of the most important enzyme involved in tumor invasion, was able to release a fragment of NC1(XIX), which retained the anti-tumor activity. Molecular modeling studies showed that NC1(XIX) and the anti-tumor fragment released by plasmin (F4) adopted locally the same type I ß-turn conformation. This suggests that the anti-tumor effect is conformation-dependent. This study demonstrates that collagen XIX is a novel proteolytic substrate for plasmin. Such release may constitute a defense of the organism against tumor invasion.

[Urinary investigations in the diagnosis and monitoring of monoclonal gammopathies in daily practice]. / Place des explorations urinaires dans le diagnostic et le suivi des gammapathies monoclonales en pratique quotidienne.

The management of monoclonal gammopathies remains a public health issue with an incidence greater than 3% of the population over 50 years. Laboratory investigations, including urinary investigations play a key role in the diagnosis and monitoring of the patients. Urinary investigations are not recommended when screening monoclonal gammopathies. However, the initial laboratory evaluation of the monoclonal gammopathies systematically relies on renal function and proteinuria assessment. Urinary proteins electrophoresis combined with urinary proteins immunofixation are also recommended in the initial evaluation, with the exception of the Waldenström's disease. In some cases, serum investigations remain negative whereas urinary investigations confirm the presence of a monoclonal component. National and international recommendations have also been published about the monitoring of monoclonal gammopathies. The biological monitoring of monoclonal gammopathy of undetermined significance is mostly done by serum tests. Urinary investigations are commonly included in the response criteria in case of multiple myeloma or AL amyloidosis. Laboratory investigations like serum free light chain assay tend to decrease the need of urinary investigations in the monoclonal gammopathies. However, these urinary investigations currently maintain a leading role in the diagnosis and monitoring of monoclonal gammopathies.

Matrikines from basement membrane collagens: a new anti-cancer strategy.

BACKGROUND: Tumor microenvironment is a complex system composed of a largely altered extracellular matrix with different cell types that determine angiogenic responses and tumor progression. Upon the influence of hypoxia, tumor cells secrete cytokines that activate stromal cells to produce proteases and angiogenic factors. In addition to stromal ECM breakdown, proteases exert various pro- or anti-tumorigenic functions and participate in the release of various ECM fragments, named matrikines or matricryptins, capable to act as endogenous angiogenesis inhibitors and to limit tumor progression. SCOPE OF REVIEW: We will focus on the matrikines derived from the NC1 domains of the different constitutive chains of basement membrane-associated collagens and mainly collagen IV. MAJOR CONCLUSIONS: The putative targets of the matrikine control are the proliferation and invasive properties of tumor or inflammatory cells, and the angiogenic and lymphangiogenic responses. Collagen-derived matrikines such as canstatin, tumstatin or tetrastatin for example, decrease tumor growth in various cancer models. Their anti-cancer activities comprise anti-proliferative effects on tumor or endothelial cells by induction of apoptosis or cell cycle blockade and the induction of a loss of their migratory phenotype. They were used in various preclinical therapeutic strategies: i) induction of their overexpression by cancer cells or by the host cells, ii) use of recombinant proteins or synthetic peptides or structural analogues designed from the structure of the active sequences, iii) used in combined therapies with conventional chemotherapy or radiotherapy. GENERAL SIGNIFICANCE: Collagen-derived matrikines strongly inhibited tumor growth in many preclinical cancer models in mouse. They constitute a new family of anti-cancer agents able to limit cancer progression. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.