Case report: First evidence of impressive efficacy of modulated dose selpercatinib in a young Caucasian with ANK3-RET fusion-positive NSCLC.

Over the past decade, molecular characterization has led to change the management of advanced non-small cell lung cancer (NSCLC) harboring driver mutations. Rearranged during transfection (RET) gene fusions, occurring in 1% to 2% of NSCLC, have emerged as an oncogenic druggable target. Systemic targeted therapies with highly selective RET inhibitors (RETi), selpercatinib and pralsetinib, represent a recent clinical breakthrough. While the development of RETi has improved survival, with their increasing use, it is crucial to be aware of the risks of rare but serious adverse events (AEs). A particular challenge for clinicians in applying targeted therapies is not only diagnosing but also interpreting rare mutations. Herein, we report a case of a 43-year-old Caucasian advanced NSCLC patient diagnosed with a rare RET gene fusion, ANK3::RET, identified with Next Generation Sequencing (NGS). Selpercatinib has been initiated at the recommended initial dose after one incomplete chemotherapy cycle due to a severe infusion reaction, but it subsequently required a dose adjustment following grade 3 (G3) AEs. During treatment, we used a particular selpercatinib dosage (160 mg in the morning and 80 mg in the evening) with good tolerance and without compromising effectiveness. Our finding broadens the range of RET fusion types in not-Asian NSCLC. To the best of our knowledge, our case demonstrates, for the first time, a clinical and radiological response to frontline highly selective RETi selpercatinib, expanding the spectrum of potential oncogenic RET fusion partners in newly diagnosed NSCLC patients. Furthermore, to our knowledge, this is the first case describing a RET fusion-positive (RET+) NSCLC patient treated with a modified selpercatinib dosage outside the drug data sheet and demonstrating a safe and effective use.

EMILIN-1 deficiency promotes chronic inflammatory disease through TGFß signaling alteration and impairment of the gC1q/α4ß1 integrin interaction.

Alterations in extracellular matrix (ECM) components that modulate inflammatory cell behavior have been shown to serve as early starters for multifactorial diseases such as fibrosis and cancer. Here, we demonstrated that loss of the ECM glycoprotein EMILIN-1 alters the inflammatory context in skin during IMQ-induced psoriasis, a disease characterized by a prominent inflammatory infiltrate and alteration of vessels that appear dilated and tortuous. Abrogation of EMILIN-1 expression or expression of the EMILIN-1 mutant E933A impairs macrophage polarization and leads to imbalanced tissue homeostasis. We found that EMILIN-1 deficiency is associated with dilated lymphatic vessels, increased macrophage recruitment and psoriasis severity. Importantly, the null or mutant EMILIN-1 background was characterized by the induction of a myofibroblast phenotype, which in turn drove macrophages towards the M1 phenotype. By using the transgenic mouse model carrying the E933A mutation in the gC1q domain of EMILIN-1, which abolishes the interaction with α4- and α9-integrins, we demonstrated that the observed changes in TGFß signaling were due to both the EMI and gC1q domains of EMILIN-1. gC1q may exert multiple functions in psoriasis, in the context of a final, more consistent inflammatory condition by controlling skin homeostasis via interaction with both keratinocytes and fibroblasts, influencing non-canonical TGFß signaling, and likely acting on lymphatic vessel structure and function. The analyses of human psoriatic lesions, in which lower levels of EMILIN-1 were present with a very rare association with lymphatic vessels, support the multifaceted role of this ECM component in the skin inflammatory scenario.

Colorectal cancer development is affected by the ECM molecule EMILIN-2 hinging on macrophage polarization via the TLR-4/MyD88 pathway.

BACKGROUND: Colorectal cancer is one of the most frequent and deadly tumors. Among the key regulators of CRC growth and progression, the microenvironment has emerged as a crucial player and as a possible route for the development of new therapeutic opportunities. More specifically, the extracellular matrix acts directly on cancer cells and indirectly affecting the behavior of stromal and inflammatory cells, as well as the bioavailability of growth factors. Among the ECM molecules, EMILIN-2 is frequently down-regulated by methylation in CRC and the purpose of this study was to verify the impact of EMILIN-2 loss in CRC development and its possible value as a prognostic biomarker. METHODS: The AOM/DSS CRC protocol was applied to Emilin-2 null and wild type mice. Tumor development was monitored by endoscopy, the molecular analyses performed by IHC, IF and WB and the immune subpopulations characterized by flow cytometry. Ex vivo cultures of monocyte/macrophages from the murine models were used to verify the molecular pathways. Publicly available datasets were exploited to determine the CRC patients' expression profile; Spearman's correlation analyses and Cox regression were applied to evaluate the association with the inflammatory response; the clinical outcome was predicted by Kaplan-Meier survival curves. Pearson correlation analyses were also applied to a cohort of patients enrolled in our Institute. RESULTS: In preclinical settings, loss of EMILIN-2 associated with an increased number of tumor lesions upon AOM/DSS treatment. In addition, in the early stages of the disease, the Emilin-2 knockout mice displayed a myeloid-derived suppressor cells-rich infiltrate. Instead, in the late stages, lack of EMILIN-2 associated with a decreased number of M1 macrophages, resulting in a higher percentage of the tumor-promoting M2 macrophages. Mechanistically, EMILIN-2 triggered the activation of the Toll-like Receptor 4/MyD88/NF-κB pathway, instrumental for the polarization of macrophages towards the M1 phenotype. Accordingly, dataset and immunofluorescence analyses indicated that low EMILIN-2 expression levels correlated with an increased M2/M1 ratio and with poor CRC patients' prognosis. CONCLUSIONS: These novel results indicate that EMILIN-2 is a key regulator of the tumor-associated inflammatory environment and may represent a promising prognostic biomarker for CRC patients.

Elastin MIcrofibriL INterfacer1 (EMILIN-1) is an alternative prosurvival VLA-4 ligand in chronic lymphocytic leukemia.

CD49d, the α4 chain of the VLA-4 integrin, is a negative prognosticator in chronic lymphocytic leukemia (CLL) with a key role in CLL cell-microenvironment interactions mainly occurring via its ligands VCAM-1 and fibronectin. In the present study, we focused on EMILIN-1 (Elastin-MIcrofibriL-INterfacer-1), an alternative VLA-4 ligand whose role has been so far reported only in non-hematological settings, by investigating: i) the distribution of EMILIN-1 in CLL-involved tissues; ii) the capability of EMILIN-1 to operate, via its globular C1q (gC1q) domain, as additional adhesion ligand in CLL; iii) the functional meaning of EMILIN-1 gC1q/VLA-4 interactions in CLL. EMILIN-1 is widely present in the CLL-involved areas of bone marrow biopsies (BMBs) without difference between CD49d negative and positive cases, displaying at least three different expression patterns: "fibrillar", "dot-like" and "mixed". The lack in CLL-BMB of neutrophil elastase, whose proteolytic activity degrades EMILIN-1 and impairs EMILIN-1 function, suggests full functional EMILIN-1 in CLL independently of its expression pattern. Functionally, EMILIN-1 gC1q domain promotes adhesion of CLL cells through specific interaction with VLA-4, and releases pro-survival signals for CLL cells, as demonstrated by enhanced ERK and AKT phosphorylation and impairment of in-vitro-induced apoptosis. EMILIN-1/VLA-4 interaction can efficiently contribute to the maintenance of the neoplastic clone in CLL.

Acquired EGFR C797G Mutation Detected by Liquid Biopsy as Resistance Mechanism After Treatment With Osimertinib: A Case Report.

BACKGROUND: Osimertinib is a third-generation EGFR-tyrosine kinase inhibitor approved for the treatment of T790M-positive non-small-cell lung cancer. More recently, osimertinib demonstrated improved disease control compared to other EGFR-TKIs. Multiple mechanisms of resistance have been described in T790M-positive patients who experienced treatment failure with osimertinib. CASE REPORT: We report the case of a 78-year-old non-smoker woman with stage IV EGFR L858R-positive lung adenocarcinoma presented with T790M mutation after five years of treatment with gefitinib. The patient was started on osimertinib, but after two and a half years of treatment experienced disease progression. The analyses of circulating tumor DNA using next-generation sequencing showed, together with the pre-existing T790M and exon 21 L858R, the presence of the EGFR C797G resistance mutation. CONCLUSION: Our case report revealed a rare EGFR-dependent acquired resistance mutation to osimertinib in circulating tumor DNA. Liquid biopsy appears to be a promising resource to understand the biology of osimertinib resistance by clonal evolution monitoring and the identification of novel resistance mechanisms.

Multimerin-2 orchestrates the cross-talk between endothelial cells and pericytes: A mechanism to maintain vascular stability.

Tumor angiogenesis is vital for the growth and development of various solid cancers and as such is a valid and promising therapeutic target. Unfortunately, the use of the currently available anti-angiogenic drugs increases the progression-free survival by only a few months. Conversely, targeting angiogenesis to prompt both vessel reduction and normalization, has been recently viewed as a promising approach to improve therapeutic efficacy. As a double-edged sword, this line of attack may on one side halt tumor growth as a consequence of the reduction of nutrients and oxygen supplied to the tumor cells, and on the other side improve drug delivery and, hence, efficacy. Thus, it is of upmost importance to better characterize the mechanisms regulating vascular stability. In this context, recruitment of pericytes along the blood vessels is crucial to their maturation and stabilization. As the extracellular matrix molecule Multimerin-2 is secreted by endothelial cells and deposited also in juxtaposition between endothelial cells and pericytes, we explored Multimerin-2 role in the cross-talk between the two cell types. We discovered that Multimerin-2 is an adhesion substrate for pericytes. Interestingly, and consistent with the notion that Multimerin-2 is a homeostatic molecule deposited in the later stages of vessel formation, we found that the interaction between endothelial cells and pericytes promoted the expression of Multimerin-2. Furthermore, we found that Multimerin-2 modulated the expression of key cytokines both in endothelial cells and pericytes. Collectively, our findings posit Multimerin-2 as a key molecule in the cross-talk between endothelial cells and pericytes and suggest that the expression of this glycoprotein is required to maintain vascular stability.

Inactivation of EMILIN-1 by Proteolysis and Secretion in Small Extracellular Vesicles Favors Melanoma Progression and Metastasis.

Several studies have demonstrated that melanoma-derived extracellular vesicles (EVs) are involved in lymph node metastasis; however, the molecular mechanisms involved are not completely defined. Here, we found that EMILIN-1 is proteolyzed and secreted in small EVs (sEVs) as a novel mechanism to reduce its intracellular levels favoring metastasis in mouse melanoma lymph node metastatic cells. Interestingly, we observed that EMILIN-1 has intrinsic tumor and metastasis suppressive-like properties reducing effective migration, cell viability, primary tumor growth, and metastasis. Overall, our analysis suggests that the inactivation of EMILIN-1 by proteolysis and secretion in sEVs reduce its intrinsic tumor suppressive activities in melanoma favoring tumor progression and metastasis.

Role of Extracellular Matrix in Gastrointestinal Cancer-Associated Angiogenesis.

Gastrointestinal tumors are responsible for more cancer-related fatalities than any other type of tumors, and colorectal and gastric malignancies account for a large part of these diseases. Thus, there is an urgent need to develop new therapeutic approaches to improve the patients' outcome and the tumor microenvironment is a promising arena for the development of such treatments. In fact, the nature of the microenvironment in the different gastrointestinal tracts may significantly influence not only tumor development but also the therapy response. In particular, an important microenvironmental component and a potential therapeutic target is the vasculature. In this context, the extracellular matrix is a key component exerting an active effect in all the hallmarks of cancer, including angiogenesis. Here, we summarized the current knowledge on the role of extracellular matrix in affecting endothelial cell function and intratumoral vascularization in the context of colorectal and gastric cancer. The extracellular matrix acts both directly on endothelial cells and indirectly through its remodeling and the consequent release of growth factors. We envision that a deeper understanding of the role of extracellular matrix and of its remodeling during cancer progression is of chief importance for the development of new, more efficacious, targeted therapies.

Distal motor neuropathy associated with novel EMILIN1 mutation.

Elastin microfibril interface-located proteins (EMILINs) are extracellular matrix glycoproteins implicated in elastogenesis and cell proliferation. Recently, a missense mutation in the EMILIN1 gene has been associated with autosomal dominant connective tissue disorder and motor-sensory neuropathy in a single family. We identified by whole exome sequencing a novel heterozygous EMILIN1 mutation c.748C>T [p.R250C] located in the coiled coil forming region of the protein, in four affected members of an autosomal dominant family presenting a distal motor neuropathy phenotype. In affected patient a sensory nerve biopsy showed slight and unspecific changes in the number and morphology of myelinated fibers. Immunofluorescence study of a motor nerve within a muscle biopsy documented the presence of EMILIN-1 in nerve structures. Skin section and skin derived fibroblasts displayed a reduced extracellular deposition of EMILIN-1 protein with a disorganized network of poorly ramified fibers in comparison with controls. Downregulation of emilin1a in zebrafish displayed developmental delay, locomotion defects, and abnormal axonal arborization from spinal cord motor neurons. The phenotype was complemented by wild-type zebrafish emilin1a, and partially the human wild-type EMILIN1 cRNA, but not by the cRNA harboring the novel c.748C>T [p.R250C]. These data suggest a role of EMILIN-1 in the pathogenesis of diseases affecting the peripheral nervous system.

Deregulated expression of Elastin Microfibril Interfacer 2 (EMILIN2) in gastric cancer affects tumor growth and angiogenesis.

Gastric cancer is a frequent human tumor and often a lethal disease. Targeted therapy for gastric carcinomas is far behind vis-à-vis other solid tumors, primarily because of the paucity of cancer-driving mutations that could be efficiently and specifically targeted by current therapy. Thus, there is a need to discover actionable pathways/proteins and new diagnostic and prognostic biomarkers. In this study, we explored the role of the extracellular matrix glycoprotein EMILIN2, Elastin Microfibril Interfacer 2, in a cohort of gastric cancer patients. We discovered that EMILIN2 expression was consistently suppressed in gastric cancer and high expression levels of this glycoprotein were linked to abnormal vascular density. Furthermore, we found that EMILIN2 had a dual effect on gastric carcinoma cells: on one hand, it decreased tumor cell proliferation by triggering apoptosis, and on the other hand, it evoked the production of a number of cytokines involved in angiogenesis and inflammation, such as IL-8. Collectively, our findings posit EMILIN2 as an important onco-regulator exerting pleiotropic effects on the gastric cancer microenvironment.

Multimerin-2 maintains vascular stability and permeability.

Multimerin-2 is an extracellular matrix glycoprotein and member of the elastin microfibril interface-located (EMILIN) family of proteins. Multimerin-2 is deposited along blood vessels and we previously demonstrated that it regulates the VEGFA/VEGFR2 signaling axis and angiogenesis. However, its role in modulating vascular homeostasis remains largely unexplored. Here we identified Multimerin-2 as a key molecule required to maintain vascular stability. RNAi knockdown of Multimerin-2 in endothelial cells led to cell-cell junctional instability and increased permeability. Mechanistically cell-cell junction dismantlement occurred through the phosphorylation of VEGFR2 at Tyr951, activation of Src and phosphorylation of VE-cadherin. To provide an in vivo validation for these in vitro effects, we generated Multimerin-2-/- (Mmrn2-/-) mice. Although Mmrn2-/- mice developed normally and displayed no gross abnormalities, endothelial cells displayed cell junctional defects associated with increased levels of VEGFR2 phospho-Tyr949 (the murine counterpart of human Tyr951), impaired pericyte recruitment and increased vascular leakage. Of note, tumor associated vessels were defective in Mmrn2-/- mice, with increased number of small and often collapsed vessels, concurrent with a significant depletion of pericytic coverage. Consequently, the Mmrn2-/- vessels were less perfused and leakier, leading to increased tumor hypoxia. Chemotherapy efficacy was markedly impaired in Mmrn2-/- mice and this was associated with poor drug delivery to the tumor xenografts. Collectively, our findings demonstrate that Multimerin-2 is required for proper vessel homeostasis and stabilization, and unveil the possibility to utilize expression levels of this glycoprotein in predicting chemotherapy efficacy.

Abrogation of EMILIN1-ß1 integrin interaction promotes experimental colitis and colon carcinogenesis.

Colon cancer is one of the first tumor types where a functional link between inflammation and tumor onset has been described; however, the microenvironmental cues affecting colon cancer progression are poorly understood. Here we demonstrate that the expression of the ECM molecule EMILIN-1 halts the development of AOM-DSS induced tumors. In fact, upon AOM-DSS treatment the Emilin1-/- (E1-/-) mice were characterized by a higher tumor incidence, bigger adenomas and less survival. Similar results were obtained with the E933A EMILIN-1 (E1-E933A) transgenic mouse model, expressing a mutant EMILIN-1 unable to interact with α4/α9ß1 integrins. Interestingly, upon chronic treatment with DSS, E1-/- and E1-E933A mice were characterized by the presence of increased inflammatory infiltrates, higher colitis scores and more severe mucosal injury respect to the wild type (E1+/+) mice. Since alterations of the intestinal lymphatic network are a well-established feature of human inflammatory bowel disease and EMILIN-1 is a key structural element in the maintenance of the integrity of lymphatic vessels, we assessed the lymphatic vasculature in this context. The analyses revealed that both E1-/- and E1-E933A mice displayed a higher density of LYVE-1 positive vessels; however, their functionality was severely compromised after colitis induction. Taken together, these results suggest that the loss of EMILIN-1 expression may cause the reduction of the inflammatory resolution during colon cancer progression due to a decreased lymph flow and impaired inflammatory cell drainage.

Multiplex staining depicts the immune infiltrate in colitis-induced colon cancer model.

Assessment of the host immune response pattern is of increasing importance as highly prognostic and diagnostic, in immune-related diseases and in some types of cancer. Chronic inflammation is a major hallmark in colon cancer formation, but, despite the extent of local inflammatory infiltrate has been demonstrated to be extremely informative, its evaluation is not routinely assessed due to the complexity and limitations of classical immunohistochemistry (IHC). In the last years, technological advance helped in bypassing technical limits, setting up multiplex IHC (mIHC) based on tyramide signal amplification (TSA) method and designing software suited to aid pathologists in cell scoring analysis. Several studies verified the efficacy of this method, but they were restricted to the analysis of human samples. In the era of translational medicine the use of animal models to depict human pathologies, in a more complete and complex approach, is really crucial. Nevertheless, the optimization and validation of this method to species other than human is still poor. We took advantage of Multispectral Imaging System to identify the immunoprofile of Dextran Sulphate Sodium (DSS)-treated mouse colon. We optimized a protocol to sequentially stain formalin fixed paraffin embedded murine colon samples for CD3, CD8a, CD4, and CD4R5B0 antigens. With this approach we obtained a detailed lymphocyte profile, while preserving the morphological tissue context, generally lost with techniques like gene expression profiling or flow cytometry. This study, comparing the results obtained by mIHC with immunophenotyping performed with cytofluorimetric and standard IHC methods validates the potentiality and the applicability of this innovative approach.

The Probe Based Confocal Laser Endomicroscopy (pCLE) in Locally Advanced Gastric Cancer: A Powerful Technique for Real-Time Analysis of Vasculature.

Probe based confocal laser endomicroscopy (pCLE) is an advanced technique which provides imaging of gastrointestinal mucosa at subcellular resolution and, importantly, a valid tool for the evaluation of microvasculature during endoscopic examination. In order to assess intratumoral vascularization and the efficiency of blood flow in locally advanced gastric cancer, we examined 57 patients through pCLE imaging. The vascular alterations in gastric cancer were mainly characterized by leakage and by the presence of tortuous and large size vessels. Defects in blood flow were detected very rarely. No association between the angiogenic score and the gastric tumor site or histological type was observed. Interestingly, no correlation was also found with the tumor grading indicating that the vascular angiogenic anomalies in gastric cancer represent an early pathological event to be observed and detected. The majority of patients displayed unchanged vascular alterations following neoadjuvant chemotherapy and this positively correlated with stable or progressive disease, suggesting that an unaltered angiogenic score could per se be indicative of poor therapeutic efficacy. Different vascular parameters were evaluated by immunofluorescence using bioptic samples and the vessel density did not correlate with clinical staging, site or histologic type. Interestingly, only CD105, Multimerin-2 and GLUT1 were able to discriminate normal from tumoral gastric mucosa. Taken together, these findings indicate that functional and structural angiogenic parameters characteristic of tumor blood network were fully detectable by pCLE. Moreover, the evaluation of tumor vasculature by real-time assessment may provide useful information to achieve tailored therapeutic interventions for gastric cancer patients.

Integrin binding site within the gC1q domain orchestrates EMILIN-1-induced lymphangiogenesis.

Lymphatic vessels (LVs) play a pivotal role in the control of tissue homeostasis and also have emerged as important regulators of immunity, inflammation and tumor metastasis. EMILIN-1 is the first ECM protein identified as a structural modulator of the growth and maintenance of LV; accordingly, Emilin1-/- mice display lymphatic morphological alterations leading to functional defects as mild lymphedema, leakage and compromised lymph drainage. Many EMILIN-1 functions are exerted by the binding of its gC1q domain with the E933 residue of α4 and α9ß1 integrins. To investigate the specific regulatory role of this domain on lymphangiogenesis, we generated a transgenic mouse model expressing an E933A-mutated EMILIN-1 (E1-E933A), unable to interact with α4 or α9 integrin. The mutant resulted in abnormal LV architecture with dense, tortuous and irregular networks; moreover, the number of anchoring filaments was reduced and collector valves had aberrant narrowed structures. E933A mutation also affected lymphatic function in lymphangiography assays and made the transgenic mice more prone to lymph node metastases. The finding that the gC1q/integrin interaction is crucial for a correct lymphangiogenesis response was confirmed and reinforced by functional in vitro tubulogenesis assays. In addition, ex vivo thoracic-duct ring assays revealed that E1-E933A-derived lymphatic endothelial cells had a severe reduction in sprouting capacity and were unable to organize into capillary-like structures. All these data provide evidence that the novel "regulatory structural" role of EMILIN-1 in the lymphangiogenic process is played by the integrin binding site within its gC1q domain.

A novel murine model for arrhythmogenic cardiomyopathy points to a pathogenic role of Wnt signalling and miRNA dysregulation.

AIMS: Arrhythmogenic cardiomyopathy (AC) is one of the most common inherited cardiomyopathies, characterized by progressive fibro-fatty replacement in the myocardium. Clinically, AC manifests itself with ventricular arrhythmias, syncope, and sudden death and shows wide inter- and intra-familial variability. Among the causative genes identified so far, those encoding for the desmosomal proteins plakophilin-2 (PKP2), desmoplakin (DSP), and desmoglein-2 (DSG2) are the most commonly mutated. So far, little is known about the molecular mechanism(s) behind such a varied spectrum of phenotypes, although it has been shown that the causative mutations not only lead to structural abnormalities but also affect the miRNA profiling of cardiac tissue. Here, we aimed at studying the pathogenic effects of a nonsense mutation of the desmoglein-2 gene, both at the structural level and in terms of miRNA expression pattern. METHODS AND RESULTS: We generated transgenic mice with cardiomyocyte-specific overexpression of a FLAG-tagged human desmoglein-2 harbouring the Q558* nonsense mutation found in an AC patient. The hearts of these mice showed signs of fibrosis, decrease in desmosomal size and number, and reduction of the Wnt/ß-catenin signalling. Genome-wide RNA-Seq performed in Tg-hQ hearts and non-transgenic hearts revealed that 24 miRNAs were dysregulated in transgenic animals. Further bioinformatic analyses for selected miRNAs suggested that miR-217-5p, miR-499-5p, and miR-708-5p might be involved in the pathogenesis of the disease. CONCLUSION: Down-regulation of the canonical Wnt/ß-catenin signalling might be considered a common key event in the AC pathogenesis. We identified the miRNA signature in AC hearts, with miR-708-5p and miR-217-5p being the most up-regulated and miR-499-5p the most down-regulated miRNAs. All of them were predicted to be involved in the regulation of the Wnt/ß-catenin pathway and might reveal the potential pathophysiology mechanisms of AC, as well as be useful as therapeutic targets for the disease.

Therapeutic decision based on molecular detection of resistance mechanism in an ALK-rearranged lung cancer patient: a case report.

Background: The use of tyrosine kinase inhibitors (TKIs) of ALK is the therapy of choice for ALK-fusion patients. Unfortunately, all patients under this kind of treatment eventually develop acquired resistance through several well-known mechanisms, such as acquisition of a secondary mutation within the kinase domain, activation of a bypass signaling pathway, or a histological change like small-cell lung cancer transformation. At the time of progression, a tissue re-biopsy may give important molecular and morphological information regarding the mechanisms driving resistance to ALK TKIs. However, this procedure is not always feasible and it may not reflect the tumor heterogeneity, and therefore gives incomplete information. To overcome these drawbacks, the analysis of circulating tumor DNA (ctDNA) isolated from plasma, the so-called liquid biopsy, is emerging as a noninvasive and useful tool for detecting resistance mutations. Secondary resistance mutations are common in second-generation TKIs resistant patients and among these, Gly1202Arg (p.G1202R) emerged as the most frequent mutation. Case presentation: We have treated an ALK-positive lung adenocarcinoma patient with a sequential strategy of ALK TKIs. Patient follow-up was performed combining clinical, radiological, and molecular profiling. ctDNA was isolated from plasma and by means of ultra-deep next generation sequencing; we searched for secondary ALK resistance mutations on exons 21-25. ALK mutation Gly1202Arg (G1202R) was detected. We have documented consistency between plasma levels of G1202R mutation and radiological progression or improvement. Conclusion: Liquid biopsy appears to be a promising tool to anticipate progression and to drive the therapeutic strategy based upon ALK resistance mutations.

Loss of Multimerin-2 and EMILIN-2 Expression in Gastric Cancer Associate with Altered Angiogenesis.

Gastric cancer is a deadly tumor and a relatively common disease worldwide. Surgical resection and chemotherapy are the main clinical options to treat this type of disease, however the median overall survival rate is limited to one year. Thus, the development of new therapies is a highly necessary clinical need. Angiogenesis is a promising target for this tumor type, however clinical trials with the use of anti-angiogenic drugs have so far not met expectations. Therefore, it is important to better characterize the expression of molecules whose expression levels may impact on the efficacy of the treatments. In this study the characteristics of the gastric tumor associated blood vessels were first assessed by endomicroscopy. Next, we analyzed the expression of Multimerin-2, EMILIN-2 and EMILIN-1, three molecules of the EMI Domain ENdowed (EDEN) protein family. These molecules play important functions in the tumor microenvironment, affecting cancer progression both directly and indirectly impinging on angiogenesis and lymphangiogenesis. All the molecules were highly expressed in the normal mucosa whereas in a number of patients their expression was altered. We consider that better characterizing the gastric tumor microenvironment and the quality of the vasculature may achieve effective patient tailored therapies.

The ablation of the matricellular protein EMILIN2 causes defective vascularization due to impaired EGFR-dependent IL-8 production affecting tumor growth.

EMILIN2 is an extracellular matrix constituent playing an important role in angiogenesis; however, the underlying mechanism is unknown. Here we show that EMILIN2 promotes angiogenesis by directly binding epidermal growth factor receptor (EGFR), which enhances interleukin-8 (IL-8) production. In turn, IL-8 stimulates the proliferation and migration of vascular endothelial cells. Emilin2 null mice were generated and exhibited delayed retinal vascular development, which was rescued by the administration of the IL-8 murine ortholog MIP-2. Next, we assessed tumor growth and tumor-associated angiogenesis in these mice. Tumor cell growth in Emilin2 null mice was impaired as well as the expression of MIP-2. The vascular density of the tumors developed in Emilin2 null mice was prejudiced and vessels perfusion, as well as response to chemotherapy, decreased. Accordingly, human tumors expressing high levels of EMILIN2 were more responsive to chemotherapy. These results point at EMILIN2 as a key microenvironmental cue affecting vessel formation and unveil the possibility to develop new prognostic tools to predict chemotherapy efficacy.