In Vivo imaging reveals extracellular vesicle-mediated phenocopying of metastatic behavior.

Most cancer cells release heterogeneous populations of extracellular vesicles (EVs) containing proteins, lipids, and nucleic acids. In vitro experiments showed that EV uptake can lead to transfer of functional mRNA and altered cellular behavior. However, similar in vivo experiments remain challenging because cells that take up EVs cannot be discriminated from non-EV-receiving cells. Here, we used the Cre-LoxP system to directly identify tumor cells that take up EVs in vivo. We show that EVs released by malignant tumor cells are taken up by less malignant tumor cells located within the same and within distant tumors and that these EVs carry mRNAs involved in migration and metastasis. By intravital imaging, we show that the less malignant tumor cells that take up EVs display enhanced migratory behavior and metastatic capacity. We postulate that tumor cells locally and systemically share molecules carried by EVs in vivo and that this affects cellular behavior.

Correction to: The Present and Future of Liquid Biopsies in Non-Small Cell Lung Cancer: Combining Four Biosources for Diagnosis, Prognosis, Prediction, and Disease Monitoring.

The original version of this review article unfortunately contained a mistake in the Funding section.

Sensing of latent EBV infection through exosomal transfer of 5'pppRNA.

Complex interactions between DNA herpesviruses and host factors determine the establishment of a life-long asymptomatic latent infection. The lymphotropic Epstein-Barr virus (EBV) seems to avoid recognition by innate sensors despite massive transcription of immunostimulatory small RNAs (EBV-EBERs). Here we demonstrate that in latently infected B cells, EBER1 transcripts interact with the lupus antigen (La) ribonucleoprotein, avoiding cytoplasmic RNA sensors. However, in coculture experiments we observed that latent-infected cells trigger antiviral immunity in dendritic cells (DCs) through selective release and transfer of RNA via exosomes. In ex vivo tonsillar cultures, we observed that EBER1-loaded exosomes are preferentially captured and internalized by human plasmacytoid DCs (pDCs) that express the TIM1 phosphatidylserine receptor, a known viral- and exosomal target. Using an EBER-deficient EBV strain, enzymatic removal of 5'ppp, in vitro transcripts, and coculture experiments, we established that 5'pppEBER1 transfer via exosomes drives antiviral immunity in nonpermissive DCs. Lupus erythematosus patients suffer from elevated EBV load and activated antiviral immunity, in particular in skin lesions that are infiltrated with pDCs. We detected high levels of EBER1 RNA in such skin lesions, as well as EBV-microRNAs, but no intact EBV-DNA, linking non-cell-autonomous EBER1 presence with skin inflammation in predisposed individuals. Collectively, our studies indicate that virus-modified exosomes have a physiological role in the host-pathogen stand-off and may promote inflammatory disease.

The Present and Future of Liquid Biopsies in Non-Small Cell Lung Cancer: Combining Four Biosources for Diagnosis, Prognosis, Prediction, and Disease Monitoring.

PURPOSE OF REVIEW: Liquid biopsies have potential as tools for diagnosis, prognosis, and prediction of response to therapy. Herein, we will extensively review four liquid biosources, tumor-educated platelets (TEPs), cell-free DNA (cfDNA), circulating tumor cells (CTCs), and extracellular vesicles (EVs) and we will clarify their optimal application in non-small cell lung cancer (NSCLC) diagnosis and therapy. RECENT FINDINGS: Liquid biopsies are a minimally invasive alternative to tissue biopsies-especially important in NSCLC patients-since tumor tissue is often unavailable or insufficient for complete genetic analysis. The main advantages of liquid biopsies include the possibility for repeated sampling, the lower cost, and the fact that they can reflect the complete molecular status of the patient better than a single-site biopsy. This is specifically important for lung adenocarcinoma patients since the detection of specific genetic alterations can predict response to targeted therapies. Molecular analysis is currently cardinal for therapy decision-making and disease monitoring in lung cancer patients. Liquid biopsies can make easier our daily clinical practice and if prospectively tested and validated may serve as a means for lung cancer early detection.

Analysis of the Optimum Gain of a High-Pass L-Matching Network for Rectennas.

Rectennas, which mainly consist of an antenna, matching network, and rectifier, are used to harvest radiofrequency energy in order to power tiny sensor nodes, e.g., the nodes of the Internet of Things. This paper demonstrates for the first time, the existence of an optimum voltage gain for high-pass L-matching networks used in rectennas by deriving an analytical expression. The optimum gain is that which leads to maximum power efficiency of the rectenna. Here, apart from the L-matching network, a Schottky single-diode rectifier was used for the rectenna, which was optimized at 868 MHz for a power range from -30 dBm to -10 dBm. As the theoretical expression depends on parameters not very well-known a priori, an accurate search of the optimum gain for each power level was performed via simulations. Experimental results show remarkable power efficiencies ranging from 16% at -30 dBm to 55% at -10 dBm, which are for almost all the tested power levels the highest published in the literature for similar designs.

Sonic-hedgehog-mediated proliferation requires the localization of PKA to the cilium base.

Cerebellar granular neuronal precursors (CGNPs) proliferate in response to the mitogenic activity of Sonic hedgehog (Shh), and this proliferation is negatively regulated by activation of cAMP-dependent protein kinase (PKA). In the basal state, the PKA catalytic subunits (C-PKA) are inactive because of their association with the regulatory subunits (R-PKA). As the level of cAMP increases, it binds to R-PKA, displacing and thereby activating the C-PKA. Here we report that, in the presence of Shh, inactive C-PKA accumulates at the cilium base of proliferative CGNPs whereas removal of Shh triggers the activation of PKA at this particular location. Furthermore, we demonstrate that the anchoring of the PKA holoenzyme to the cilium base is mediated by the specific binding of the type II PKA regulatory subunit (RII-PKA) to the A-kinase anchoring proteins (AKAPs). Disruption of the interaction between RII-PKA and AKAPs inhibits Shh activity and, therefore, blocks proliferation of CGNP cultures. Collectively, these results demonstrate that the pool of PKA localized to the cilium base of CGNP plays an essential role in the integration of Shh signal transduction.

LCOR mediates interferon-independent tumor immunogenicity and responsiveness to immune-checkpoint blockade in triple-negative breast cancer.

Ligand-dependent corepressor (LCOR) mediates normal and malignant breast stem cell differentiation. Cancer stem cells (CSCs) generate phenotypic heterogeneity and drive therapy resistance, yet their role in immunotherapy is poorly understood. Here we show that immune-checkpoint blockade (ICB) therapy selects for LCORlow CSCs with reduced antigen processing/presentation machinery (APM) driving immune escape and ICB resistance in triple-negative breast cancer (TNBC). We unveil an unexpected function of LCOR as a master transcriptional activator of APM genes binding to IFN-stimulated response elements (ISREs) in an IFN signaling-independent manner. Through genetic modification of LCOR expression, we demonstrate its central role in modulation of tumor immunogenicity and ICB responsiveness. In TNBC, LCOR associates with ICB clinical response. Importantly, extracellular vesicle (EV) Lcor-messenger RNA therapy in combination with anti-PD-L1 overcame resistance and eradicated breast cancer metastasis in preclinical models. Collectively, these data support LCOR as a promising target for enhancement of ICB efficacy in TNBC, by boosting of tumor APM independently of IFN.

Cell memory of epithelial-mesenchymal plasticity in cancer.

Fundamental biological processes of cell identity and cell fate determination are controlled by complex regulatory networks. These processes require molecular mechanisms that confer cellular phenotypic memory and state persistence. In this minireview, we will summarize mechanisms of cell memory based on regulatory hysteretic feedback loops and explore epigenetic mechanisms widely represented in nature, with special focus on epithelial-to-mesenchymal plasticity. We will also discuss the functional consequences of cell memory and epithelial-to-mesenchymal plasticity dynamics during development and cancer metastasis.

SRC and PIM1 as potential co-targets to overcome resistance in MET deregulated non-small cell lung cancer.

BACKGROUND: The role of MET alterations in non-small cell lung cancer (NSCLC) is increasing and several targeted agents are under evaluation. MET exon 14 skipping mutations and MET amplifications are associated with potential sensitivity to MET inhibition, though resistance mechanisms are emerging. In MET addicted cells, MET inhibition leads to activation of proviral integration site for Moloney murine leukemia virus-1 (PIM1). PIM1 and proto-oncogene tyrosine-protein kinase Src (SRC) can regulate the expression of receptor tyrosine kinases (RTKs), potentially inducing resistance to MET inhibition through cross-activation. METHODS: We evaluated the activity of class I-II MET inhibitors, the SRC inhibitor dasatinib, and pan-PIM inhibitors in four MET addicted cell lines. We assessed the effect of the dual MET/PIM and MET/SRC inhibition on cell viability and at the protein level. We evaluated RNA expression profiles of the cell lines. Advanced NSCLCs were also screened for MET alterations. RESULTS: All cell lines were sensitive to class I-II MET inhibitors. All cell lines were resistant to single PIM and SRC inhibition. Dual MET/PIM inhibition was synergistic or additive in MET amplified cell lines and dual MET/SRC inhibition was highly synergistic in all MET addicted cell lines. The addition of an SRC inhibitor partially prevents the RTKs cross-activation. MET alterations were found in 9 out of 97 evaluable samples (9.3%); median overall survival in MET altered patients was 5 months (95% CI, 3 m-NA). CONCLUSIONS: We identified a potential role of PIM inhibition in MET amplified tumors and of SRC inhibition in MET addicted tumors. Potential applications of this new treatment strategy warrant further evaluation.

Osimertinib and pterostilbene in EGFR-mutation-positive non-small cell lung cancer (NSCLC).

Monotherapy with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) still leads to incomplete responses in most EGFR-mutation positive non-small cell lung cancer (NSCLC) patients, often due to acquired resistance through activation of parallel compensatory pathways. We have previously shown that co-targeting EGFR, signal transducer and activator of transcription 3 (STAT3), and Src-yes-associated protein 1 (YAP1) was highly synergistic in vitro and in vivo. In the present study, we treated EGFR-mutation positive cell lines with the combination of osimertinib plus a natural compound, pterostilbene, which has been reported to abrogate Src and STAT3 activation. Methods: Cell viability assays and immunoblotting were performed to reveal the mechanisms of action of pterostilbene, osimertinib and pterostilbene plus osimertinib in five EGFR-mutation positive NSCLC and one triple negative breast cancer (TNBC) cell lines. Results: Osimertinib plus pterostilbene yielded synergistic effects in all EGFR-mutation positive NSCLC cell lines investigated. Surprisingly, pterostilbene alone did not inhibit, nor downregulate Src phosphorylation in the EGFR-mutation positive NSCLC cell lines or the TNBC cell line, MDA-MB-231. However, the double combination of osimertinib plus pterostilbene reversed the osimertinib-induced STAT3, YAP1, and CUB domain-containing protein-1 (CDCP1) phosphorylation and slightly suppressed Src phosphorylation in PC9 and H1975 cells. Conclusion: The results of this study indicate that pterostilbene may be used to abrogate the activated resistance pathways of single osimertinib treatment in EGFR-mutation positive NSCLC. Future studies should focus on in vivo translation and confirmation of these results.

BRAF Mutations Classes I, II, and III in NSCLC Patients Included in the SLLIP Trial: The Need for a New Pre-Clinical Treatment Rationale.

BRAF V600 mutations have been found in 1-2% of non-small-cell lung cancer (NSCLC) patients, with Food and Drug Administration (FDA) approved treatment of dabrafenib plus trametinib and progression free survival (PFS) of 10.9 months. However, 50-80% of BRAF mutations in lung cancer are non-V600, and can be class II, with intermediate to high kinase activity and RAS independence, or class III, with impaired kinase activity, upstream signaling dependence, and consequently, sensitivity to receptor tyrosine kinase (RTK) inhibitors. Plasma cell-free DNA (cfDNA) of 185 newly diagnosed advanced lung adenocarcinoma patients (Spanish Lung Liquid versus Invasive Biopsy Program, SLLIP, NCT03248089) was examined for BRAF and other alterations with a targeted cfDNA next-generation sequencing (NGS) assay (Guardant360®, Guardant Health Inc., CA, USA), and results were correlated with patient outcome. Cell viability with single or combined RAF, MEK, and SHP2 inhibitors was assessed in cell lines with BRAF class I, II, and III mutations. Out of 185 patients, 22 had BRAF alterations (12%) of which seven patients harbored amplifications (32%) and 17 had BRAF mutations (77%). Of the BRAF mutations, four out of 22 (18%) were V600E and 18/22 (82%) were non-V600. In vitro results confirmed sensitivity of class III and resistance of class I and II BRAF mutations, and BRAF wild type cells to SHP2 inhibition. Concomitant MEK or RAF and SHP2 inhibition showed synergistic effects, especially in the class III BRAF-mutant cell line. Our study indicates that the class of the BRAF mutation may have clinical implications and therefore should be defined in the clinical practice and used to guide therapeutic decisions.

Hsp90 inhibitors enhance the antitumoral effect of osimertinib in parental and osimertinib-resistant non-small cell lung cancer cell lines.

BACKGROUND: Osimertinib improve therapy for non-small cell lung cancer (NSCLC). However, invariable acquired resistance appears. METHODS: MTT assay was used to analyze cell viability. Protein expression and activation was detected by Western blotting. In addition, the effects of heat shock protein 90 (Hsp90) inhibitors and osimertinib were studied in colony formation assays. RESULTS: Our laboratory generated osimertinib resistant cell lines from PC9 cell line and overexpression or activation of several proteins was detected. Hsp90 inhibitors, ganetespib and luminespib, inhibited cell viability and colony formation in H1975, PC9 and PC9-derived osimertinib-resistant cell lines and combination of these inhibitors with osimertinib achieved to enhance this cell viability and colony formation inhibition. Luminespib downregulated the expression of the several proteins involved in osimertinib-resistance and the combination of this compound plus osimertinib caused an important decrease of expression of several of these proteins, such as Stat3, Yap, Akt, EGFR and Met. Osimertinib activated the phosphorylation of several membrane receptors and downstream molecules that was partially inhibited by luminespib. In addition, a lung cancer patient with an EGFR eon 20 mutation had a partial radiographic response to ganetespib. CONCLUSIONS: Hsp90 inhibitors and osimertinib exhibits a good efficiency to inhibit cell viability, colony formation and inhibits expression and activation of proteins involved in osimertinib-resistance and may represent an effective strategy for NSCLC with intrinsic resistance to osimertinib inhibition.

Cancer Stem Cell Biomarkers in EGFR-Mutation-Positive Non-Small-Cell Lung Cancer.

INTRODUCTION: Epidermal growth factor receptor (EGFR) pathway deregulation promotes the acquisition of stemlike properties in non-small-cell lung cancer. EGFR inhibition through NOTCH enriches lung cancer stem cells (CSCs). Src through Yes-associated protein 1 (YAP1) activates NOTCH. Signal transduction and activator of transcription 3 (STAT3) activation occurs upon EGFR blockade and regulates the generation of CSCs. PATIENTS AND METHODS: Using the Aldefluor assay kit, we investigated the enrichment of aldehyde dehydrogenase (ALDH)-positive cells in EGFR-mutation-positive cells treated with gefitinib, afatinib, and osimertinib. Western blot analysis was performed to evaluate changes in CSC marker expression upon EGFR blockade. We performed gene expression analysis in a cohort of EGFR-mutation-positive non-small-cell lung cancer patients. We evaluated the association of gene expression with treatment outcomes. RESULTS: The cell subpopulation surviving EGFR inhibition had high ALDH activity and elevated CSC marker expression. Concurrent inhibition of EGFR, STAT3, and Src diminished the CSC subpopulation in an EGFR-mutation-positive cellular model. In a cohort of 64 EGFR-mutation-positive patients, 2 ALDH1 isoforms and the NOTCH target hairy and enhancer of split 1 (HES1), when highly expressed, were predictive of worse outcome to EGFR blockade. The gene expression of B-cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1) that maintains the self-renewal of stem cells was also related to treatment outcome. CONCLUSION: Single EGFR inhibitors increase the population of CSCs. Combinatory therapy targeting STAT3 and Src may be of potential benefit. ALDH1, HES1, and Bmi-1 are essential biomarkers in the initial assessment of EGFR-mutation-positive patients.

Integrin-linked kinase (ILK) and src homology 2 domain-containing phosphatase 2 (SHP2): Novel targets in EGFR-mutation positive non-small cell lung cancer (NSCLC).

BACKGROUND: The activation of multiple signaling pathways jeopardizes the clinical efficacy of EGFR tyrosine kinase inhibitors (TKIs) in EGFR-mutation positive non-small cell lung cancer (NSCLC). Integrin-linked kinase (ILK) regulates the interactions between tumor cells and extracellular environment to activate signaling pathways and promote cell proliferation, migration, and epithelial-mesenchymal transition. Src homology 2 domain-containing phosphatase 2 (SHP2) is essential for receptor tyrosine kinase signaling and mitogen-activated protein kinase (MAPK) pathway activation. METHODS: We analyzed tumor ILK, ß-receptor subunit glycoprotein 130 (gp130), SHP2, and stromal hepatocyte growth factor (HGF) and interleukin-6 (IL-6) mRNA expression in baseline tumor specimens of advanced EGFR-mutation positive NSCLC patients treated with EGFR TKIs. RESULTS: ILK, when highly expressed, was an independent poor prognostic factor for the progression-free survival of the patients, both in the univariate (hazard ratio [HR for disease progression, 2.49; 95% CI, 1.37-4.52; P = .0020]) and in the multivariate (HR 3.74; 95% CI, 1.33-10.56; P = .0126) Cox regression model. Patients with high SHP2 expression had an almost 13-month shorter progression-free survival (P = .0094) and an 18-month shorter overall survival (P = .0182) in comparison to those with low SHP2 mRNA expression. INTERPRETATION: The levels of ILK and SHP2 could be predictive for upfront combinatory therapy of EGFR TKIs plus SHP2 or ILK inhibitors. FUND: A grant from La Caixa Foundation, an Instituto de Salud Carlos III grant (RESPONSE, PIE16/00011), an Instituto de Salud Carlos III grant (PI14/01678), a Marie Sklodowska-Curie Innovative Training Networks European Grant (ELBA No 765492) and a Spanish Association Against Cancer (AECC) grant (PROYE18012ROSE).

STAT3 as a potential immunotherapy biomarker in oncogene-addicted non-small cell lung cancer.

Immune checkpoint blockade has modified the treatment landscape for many types of tumors, including lung cancer. Still our knowledge on the biology of the interaction between tumor cells and the microenvironment is limited, preventing the optimal use of these new compounds and the maximum benefit that the patients can derive from them. We have actively worked on the role of STAT3, a transcriptional factor that causes innate resistance to targeted therapies in oncogene-addicted tumors. In this short review we take the opportunity to express our opinion and review existing knowledge on the immune role of STAT3 and the possible implications that this may have for the discovery of new biomarkers to predict response to immunotherapy, as well as new partners to combine with and increase the efficacy of immune checkpoint inhibitors.

Common Co-activation of AXL and CDCP1 in EGFR-mutation-positive Non-smallcell Lung Cancer Associated With Poor Prognosis.

Epidermal growth factor receptor (EGFR)-mutation-positive non-smallcell lung cancer (NSCLC) is incurable, despite high rates of response to EGFR tyrosine kinase inhibitors (TKIs). We investigated receptor tyrosine kinases (RTKs), Src family kinases and focal adhesion kinase (FAK) as genetic modifiers of innate resistance in EGFR-mutation-positive NSCLC. We performed gene expression analysis in two cohorts (Cohort 1 and Cohort 2) of EGFR-mutation-positive NSCLC patients treated with EGFR TKI. We evaluated the efficacy of gefitinib or osimertinib with the Src/FAK/Janus kinase 2 (JAK2) inhibitor, TPX0005 in vitro and in vivo. In Cohort 1, CUB domain-containing protein-1 (CDCP1) was an independent negative prognostic factor for progression-free survival (hazard ratio of 1.79, p=0.0407) and overall survival (hazard ratio of 2.23, p=0.0192). A two-gene model based on AXL and CDCP1 expression was strongly associated with the clinical outcome to EGFR TKIs, in both cohorts of patients. Our preclinical experiments revealed that several RTKs and non-RTKs, were up-regulated at baseline or after treatment with gefitinib or osimertinib. TPX-0005 plus EGFR TKI suppressed expression and activation of RTKs and downstream signaling intermediates. Co-expression of CDCP1 and AXL is often observed in EGFR-mutation-positive tumors, limiting the efficacy of EGFR TKIs. Co-treatment with EGFR TKI and TPX-0005 warrants testing.

Glycosylated extracellular vesicles released by glioblastoma cells are decorated by CCL18 allowing for cellular uptake via chemokine receptor CCR8.

Cancer cells release extracellular vesicles (EVs) that contain functional biomolecules such as RNA and proteins. EVs are transferred to recipient cancer cells and can promote tumour progression and therapy resistance. Through RNAi screening, we identified a novel EV uptake mechanism involving a triple interaction between the chemokine receptor CCR8 on the cells, glycans exposed on EVs and the soluble ligand CCL18. This ligand acts as bridging molecule, connecting EVs to cancer cells. We show that glioblastoma EVs promote cell proliferation and resistance to the alkylating agent temozolomide (TMZ). Using in vitro and in vivo stem-like glioblastoma models, we demonstrate that EV-induced phenotypes are neutralised by a small molecule CCR8 inhibitor, R243. Interference with chemokine receptors may offer therapeutic opportunities against EV-mediated cross-talk in glioblastoma.

The combination of checkpoint immunotherapy and targeted therapy in cancer.

The therapeutic possibilities for patients with metastatic melanoma have changed due to the development of targeted therapies that inhibit oncogenic signaling pathways as well as immune modulating therapies that unleash the patient antitumor immunity. These therapeutic changes have impressively increased the median overall survival of the patients. Considering the dramatic but transient responses that occur with targeted therapies for a subgroup of patients and the durable responses that can be achieved with immunotherapy in a subset of patients, a lot of effort is ongoing for the clinical development of combinations of these two therapeutic approaches. Herein we discuss the existing preclinical and clinical data for the combination of targeted therapies and immunotherapy focusing mainly on melanoma and non-small cell lung cancer (NSCLC).

Anaplastic lymphoma kinase inhibitors in phase I and phase II clinical trials for non-small cell lung cancer.

INTRODUCTION: Crizotinib is a first-in-class ALK tyrosine kinase inhibitor (TKI), which has proven its superiority over standard platinum-based chemotherapy for the first-line therapy of ALK-rearranged non-small cell lung cancer (NSCLC) patients. The development of acquired resistance to crizotinib represents an ongoing challenge with the central nervous system being one of the most common sites of relapse. Ceritinib and alectinib are approved second-generation ALK TKIs. Several novel ALK inhibitors, more potent and with different selectivity compared to crizotinib, are currently in development. Areas covered: This review will focus on new ALK inhibitors, currently in phase 1 or 2 clinical studies. We will also comment on the mechanisms of resistance to ALK inhibition and the strategies to delay or overcome resistance. Expert opinion: The therapeutic management of ALK-rearranged NSCLC has been greatly improved. Next-generation ALK inhibitors have shown differential potency against ALK rearrangements and ALK resistance mutations. The molecular profile of the tumor at the time of disease progression to crizotinib is crucial for the sequencing of novel ALK TKIs. Ongoing clinical studies will address key issues, including the optimal therapeutic algorithm and whether combinational approaches are more effective than single ALK inhibition for the outcome of ALK-rearranged NSCLC patients.

Rearranged EML4-ALK fusion transcripts sequester in circulating blood platelets and enable blood-based crizotinib response monitoring in non-small-cell lung cancer.

PURPOSE: Non-small-cell lung cancers harboring EML4-ALK rearrangements are sensitive to crizotinib. However, despite initial response, most patients will eventually relapse, and monitoring EML4-ALK rearrangements over the course of treatment may help identify these patients. However, challenges associated with serial tumor biopsies have highlighted the need for blood-based assays for the monitoring of biomarkers. Platelets can sequester RNA released by tumor cells and are thus an attractive source for the non-invasive assessment of biomarkers. METHODS: EML4-ALK rearrangements were analyzed by RT-PCR in platelets and plasma isolated from blood obtained from 77 patients with non-small-cell lung cancer, 38 of whom had EML4-ALK-rearranged tumors. In a subset of 29 patients with EML4-ALK-rearranged tumors who were treated with crizotinib, EML4-ALK rearrangements in platelets were correlated with progression-free and overall survival. RESULTS: RT-PCR demonstrated 65% sensitivity and 100% specificity for the detection of EML4-ALK rearrangements in platelets. In the subset of 29 patients treated with crizotinib, progression-free survival was 3.7 months for patients with EML4-ALK+ platelets and 16 months for those with EML4-ALK- platelets (hazard ratio, 3.5; P = 0.02). Monitoring of EML4-ALK rearrangements in the platelets of one patient over a period of 30 months revealed crizotinib resistance two months prior to radiographic disease progression. CONCLUSIONS: Platelets are a valuable source for the non-invasive detection of EML4-ALK rearrangements and may prove useful for predicting and monitoring outcome to crizotinib, thereby improving clinical decisions based on radiographic imaging alone.