Treatment resistance and metastasis are linked to cancer stem cells (CSCs). This population represents a promising target, but remains unexplored in lung cancer. The main objective of this study was to characterize lung CSCs and discover new therapeutic strategies

Amado, H.

Blasco, Ana

Calabuig, Silvia

Camps, C.

Durendez-Saez, Elena

Escorihuela, Eva

Herreros Pomares, Alejandro

Jantus Lewintre, Eloisa

Murga, Juan

Navarro, A.

Sampedro, C.

Torres, Susana

Zhang, F.

Annals of Oncology

English

Repositori Institucional de la Universitat Jaume I

maximization method was used to identify a SIP cut-off level and dichotomize ptsaccordingly. The objective was to correlate SIP with clinical characteristics and RECISTresponse by univariate logistic regression analysis.Results: 37 aNSCLC pts were evaluable for SIP before IO: 32% 65 years, 91% non-squamous, 43%KRASmutated, 51%with PD-L1 expression1%, 8% chemotherapynaı¨ve. 43% had PD, 41% stability (SD), 16% partial response (PR). Median PFS and OSwere 2.7 (95%CI 1.8; 7.3) and 13 (95%CI 4.8-NR)months, respectively, medianfollow-up was 9.3 (95% CI 6.2-14.9) months. SIP (%CD28-CD57þKLRG1þ) medianvalue on circulating CD8þ lymphocytes was 12.2% (min 1.7%, max 56.1%). 32% of ptshad>20.47%CD8þ lymphocytes with a CD28-CD57þKLRG1þ phenotype, being clas-sified SIPþ. SIP status did not significantly correlate with age, pts’ characteristics orCT exposure. 2 (17%) of 12 SIPþ had PR/SD (DCR), vs 19 (76%) of 25 SIP- pts(p¼ 0.001); median PFS was significantly lower in SIPþ (1.5 months 95%CI 1;2.2) vsSIP- pts (7.4 months 95%CI 5.5, 9.3) (p¼ 0.001). Among 61 aNSCLC pts treated with1st line PCT, 18% had PD, 43% SD, 39% PR. SIP median value on circulating CD8þlymphocytes was 17.9% (min 0.89%,max 66.1%), 43% of pts were SIPþ. SIP did notsignificantly correlate with DCR (OR: 0.82, 95% CI 0.22-3.13, p¼ 0.82) upon PCT.Conclusions: iSenescence, monitored by FCmeasurement of 3 surface molecules oncirculating CD8 þ lymphocytes, is observed in 32% and 43% of aNSCLC pts before IOor PCT, respectively. SIP correlated with lower DCR upon IO and not PCT.Legal entity responsible for the study: Institut Gustave Roussy.Funding:Has not received any funding.Disclosure: All authors have declared no conflicts of interest.24P A new bioinformatic pipeline allows the design of small, targeted genepanels for efficient TMB estimationP. Manca1, I. Mallona2, D. Santini1, G. Tonini1, C.D. Rolfo3, M.D. Robinson2, F. Pantano11Medical Oncology Department, Campus Bio-Medico University, Rome, Italy, 2Instituteof Molecular Life Sciences, University of Zurich, Zurich, Switzerland, 3Thoracic Oncology& Early Clinical Trials, Medicine Department, University of Maryland Marlene andStewart Greenebaum Comprehensive Cancer Center, Baltimore, MD, USABackground: The tumor mutation burden (TMB) is emerging as a prognostic and pre-dictive marker for the response to immune checkpoint blockade (ICB) drugs. Weaimed to develop a newmethod for the definition of gene panels that can precisely esti-mate the TMB with a considerably lower amount of genome.Methods:We developed a bioinformatic pipeline which allows the design of gene pan-els suited for TMB estimation. The method is particularly efficient in optimizing thebalance between the precision of the TMB estimate and the length of the gene panel cre-ated. We tested in silico the efficiency of different panels obtained with ourmethod inan independent cohort of patients with lung adenocarcinoma (LUAD).We also com-pared in the same cohort of patients the performance of our panels with the perform-ance of existing gene panels.Results:We designed a 0.080Megabases (Mb) long gene panel which estimated TMBin an independent LUAD cohort with an acceptable precision (adjusted R2¼0.745;Spearman q¼ 0.827, Pearson q¼ 0.864). The panel showed 0.89 accuracy in the identi-fication of TMB-high patients (25/28 patients, CI: 0.73 – 0.96). Every unitary increaseof our TMB estimate was associated with lower risk of disease progression (univariateanalysis: HR¼ 0.78; CI: 0.65-0.93; p¼ 0.006; multivariate analysis: HR¼ 0.8; CI: 0.63-1.01; p¼ 0.0621). Different existing panels of less than 1Mb long showed a loweradjusted R2 when compared to our gene panel (Table). Two other commercial panelsof 1.9Mb and 1.1Mb showed a similar adjusted R2 to panels of the same lengths builtwith our method; nevertheless, they showed a lower accuracy in TMB-high patientsdefinition (ROC curves AUC of 0.862, 0.870, 0.946 and 0.967 were observed, respec-tively, for the commercial 1.9Mb panel, the commercial 1.1Mb panel, our 0.080Mbpanel and our 2.0Mb panel).Conclusions:Ourmethod allows the design of gene panels particularly suitable for theTMB estimation in patients with LUAD and outperforms existing gene panels less than1Mb long.Legal entity responsible for the study: PaoloManca.Funding:Has not received any funding.Disclosure: All authors have declared no conflicts of interest.25P Lung tumorspheres as a drug screening platform against cancer stemcellsA. Herreros Pomares1, H. Amado2, S. Calabuig Fari~nas3, E. Escorihuela1, J. Murga4,S. Torres2, E. Durendez-Saez5, F. Zhang2, A. Blasco6, A. Navarro7, C. Sampedro8,E. Jantus-Lewintre9, C. Camps101Molecular Oncology Laboratory-CIBERONC, Fundacion de Investigacion HospitalGeneral Universitario de Valencia, Valencia, Spain, 2Molecular Oncology Laboratory,Fundacion de Investigacion Hospital General Universitario de Valencia, Valencia, Spain,3Departamento de Patologıa, Universitat de Vale`ncia, Fundacion de InvestigacionHospital General Universitario de Valencia Molecular Oncology Laboratory-CIBERONC,Valencia, Spain, 4Departamento de Quımica Inorganica y Organica, Universitat JaumeI, Castellon, Spain, 5Molecular Oncology Lab, Fundacion de Investigacion HospitalGeneral Universitario de Valencia, Valencia, Spain, 6Servicio de Oncologıa Me´dica,Hospital General Universitario de Valencia, Fundacion de Investigacion HospitalGeneral Universitario de Valencia Molecular Oncology Laboratory-CIBERONC, Valencia,Spain, 7Servicio de Anatomıa Patologica, Hospital General Universitario Valencia,Valencia, Spain, 8Servicio de Cirugıa Toracica, Hospital General Universitario Valencia,Valencia, Spain, 9Departamento de Biotecnologıa, Universitat Polite`cnica de Vale`ncia,Fundacion de Investigacion Hospital General Universitario de Valencia MolecularOncology Laboratory-CIBERONC, Valencia, Spain, 10Servicio de Oncologıa Me´dica,Hospital General Universitario de Valencia, Departamento de Medicina, Universitat deVale`ncia, Fundacion de Investigacion Hospital General Universitario de ValenciaMolecular Oncology Laboratory-CIBERONC, Valencia, SpainBackground: Treatment resistance andmetastasis are linked to cancer stem cells(CSCs). This population represents a promising target, but remains unexplored in lungcancer. Themain objective of this study was to characterize lung CSCs and discovernew therapeutic strategies.Methods: The study was performed on NSCLC cells from 8 resected patients and 12cell lines. Suspension cultures (tumorspheres) were established for CSCs enrichmentand differentiated tumor cells were cultured as monolayers (2D). The CSCs propertiesof tumorspheres were assessed in vitro and in vivo. The expression of 60 CSC-relatedgenes was analyzed by RTqPCR and the expression of 12 proteins was evaluated byimmunoblot (IB) and immunofluorescence (IF). High-throughput screening was per-formed using Prestwick andMyria libraries. Selected drugs were administered intraper-itoneally to NOD/SCIDmice with tumors induced by NSCLC patient and H1650tumorspheres.Results: Lung tumorspheres showed unlimited exponential growth (>30 passages),great tumor initiation potential, differentiation capacity, and high resistance to chemo-therapy agents, but not to salinomycin. Tumorspheres had significantly higher expres-sion of CSC-related genes (ALDH1A1, KLF4, NANOG, CD44, CD90, CDKN1A,JUNB,MDM2), invasion promoters (MMP9, SNAI1, ITGA6), and Notch (NOTCH1,NOTCH3, DLL4, JAG1) andWnt (CTNNB1, GSK3B) components than their pairedadherent-cultured cells. IB confirmed the overexpression of proteins encoded by CD44,NANOG, CDKN1A, SNAI1, ITGA6, and NOTCH3, and IF showed different localiza-tion patterns on lung adenocarcinoma tumorspheres compared with the 2D cultures.Three novel drugs [Disulfiram (DSF), Compound 1 (COMP1) and Compound 2(COMP2)] with greater cytotoxic potential against lung tumorspheres thanmonolayercells were identified. These results were validated in vivo, demonstrating the capacity ofthese drugs to reduce tumor growth in mice.Conclusions: Tumorspheres are a useful culture platform for CSCs characterization ina simple and cost-effective way.We found three drugs which are able to diminish theformation and viability of tumorspheres, constituting promising therapies against lungCSCs. Supported by CB16/12/00350, PI12-02838, and PI15-00753 from ISCIII.Legal entity responsible for the study: Fundacion de InvestigacionHospital GeneralUniversitario de Valencia.Funding: Centro de Investigacion Biome´dica en Red de Oncologıa (CIBERONC) andInstituto de Salud Carlos III (ISCIII).Disclosure: All authors have declared no conflicts of interest.Table: 24PPerformance (adjusted R2) in TMB estimation of existing gene panelsmethod of similar length.Other panelsperformanceOur panelsperformance(length)panel #1 (0.187Mb) 0.76 0.83 (0.187Mb)panel #2 (0.240Mb) 0.58 0.85 (0.25Mb)panel #3 (0.300Mb) 0.69 0.85 (0.25Mb)panel #4 (0.98Mb) 0.81 0.91 (0.98Mb)panel #5 (1.1Mb) 0.92 0.92 (1.1Mb)panel #6 (1.9Mb) 0.93 0.93 (2.0Mb)abstracts Annals of Oncologyii8 | Translational research Volume 30 | Supplement 2 | April 2019This is an Open Access article under the  CC-BY-NC license.-ND

Lung tumorspheres as a drug screening platform against cancer stem cells

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Lung tumorspheres as a drug screening platform against cancer stem cells

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