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Ruben Pio



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    MA17 - Molecular Mechanisms and Therapies (ID 143)

    • Event: WCLC 2019
    • Type: Mini Oral Session
    • Track: Biology
    • Presentations: 1
    • Now Available
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      MA17.11 - High Sensitivity to PD-1 Blockade Therapy After Ld1 Depletion in KRAS-Driven Lung Cancer Through CD8+/CD3+ Tumor Infiltration and PD-L1 Induction (Now Available) (ID 2562)

      15:45 - 17:15  |  Author(s): Ruben Pio

      • Abstract
      • Presentation
      • Slides

      Background

      PD-1/PDL-1 inhibitors are approved for non-small cell lung cancer (NSCLC). However, many patients do not benefit and therapeutic combinations are under investigation. We have previously described Id1, involved in proliferation, angiogenesis and immunosuppression, as a prognostic factor in lung adenocarcinoma (LUAD) (Ponz-Sarvise, Clin Cancer Res 2011), Id1’s role in lung cancer metastasis (Castanon, Cancer Letters 2017) and more recently shown that Id1 sustains mutant KRAS-driven progression and metastasis in NSCLC (Roman, Cancer Res 2019).

      In a previous syngeneic murine lung cancer model with depleted levels of Id1 using Id1-/- and Id1 wildtype C57BL/6 mice inoculated with Lewis Lung Carcinoma (LLC), we tested a combined therapeutic strategy targeting PD-1 and Id1, showing impaired tumor growth and increased survival (Gil-Bazo, presented at WCLC 2018).

      Here we study a combined strategy targeting PD-1 and Id1 in a KRAS-mutant murine LUAD model and the immune-related mechanisms involved.

      Method

      First, a correlation between Id1 and PD-L1 mRNA expression was studied in mutant and wild-type KRAS LUAD cohorts from The Cancer Genome Atlas data set (TCGA).

      Secondly, a syngeneic tumor model using Balb/c mice through subcutaneous injection of KRAS-mutant LUAD (Lacun3) cells and Id1-silenced Lacun3 (Id1sh) cells. In vitro, proliferation was measured in both cell lines through MTS assays. IFNg-induced PD-L1 expression in both cell lines and flow cytometry was used to evaluate its mechanistic effects on the immune response.

      After tumor cells injection, mice were treated with an anti-PD-1 (RMP-1-14) monoclonal antibody or PBS, i.p. Tumor volumes according to Id1 status in tumor cells and the treatment administered were quantified. Vectra 3.0™ multispectral microscopy was used to characterize the tumor associated immune cells in paraffin-embedded tissues from our previous syngeneic murine lung cancer model using Id1-/- and Id1 wildtype C57BL/6 mice inoculated with LLC in which the combined blockade had been reported as effective. Immune marker antibodies were used to study expression of CD3, CD4 and CD8.

      Result

      An inverse, moderate and statistically significant correlation between Id1 and PD-L1 expression in mutant and wild-type KRAS LUAD cohorts from TCGA was found in both cohorts (-0.367 and -0.351, respectively, p<0.001), indicating that Id1 depletion may lead to PD-L1 expression induction.

      In vitro assays showed that Id1 silencing reduced Lacun3 cells proliferation (p<0.001). Up-regulation of surface PD-L1 expression occurred in Id1sh cells, but not in Lacun3 cells, after receiving IFNg (p=0.0022). Mechanistically, in the syngeneic murine model, Id1 inhibition in the injected cells, combined with anti-PD-1 treatment, significantly induced a tumor growth impairment (p<0.001). An intense CD8+ and CD3+ immune cell infiltration was observed in LLC Id1-/- C57BL/6 mice treated with anti-PD1 (p<0.05 for CD3+ TILS), compared the control groups, possibly explaining the dramatic tumor growth impairment previously shown on the treated animals.

      Conclusion

      Id1 silencing may induce PD-L1 overexpression according to in silico and in vitro results. Id1 and PD-1 combined blockade in our KRAS-mutant syngeneic murine LUAD model significantly impaired tumor growth, compared to each strategy alone. A significantly increased CD3+ and CD8+ tumor infiltration and IFNg-induced PD-L1 tumor expression after the combined blockade may explain these findings.

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    MS18 - Role of Biomarkers in Lung Cancer Screening (ID 81)

    • Event: WCLC 2019
    • Type: Mini Symposium
    • Track: Screening and Early Detection
    • Presentations: 1
    • Now Available
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      MS18.04 - Alternative and Promising Biomarkers (Now Available) (ID 3547)

      14:30 - 16:00  |  Presenting Author(s): Ruben Pio

      • Abstract
      • Presentation
      • Slides

      Abstract

      Randomized controlled trials have demonstrated that lung cancer screening with low-dose computed tomography (LDCT) in subjects at risk is associated with a decrease in mortality (1-3). However, concerns regarding false positive findings, overdiagnosis, or selection criteria may limit their implementation and sustainability. The use of molecular biomarkers that help to overcome some of these limitations offers great potential. Biomarkers should be non-invasive, reproducible and improve the current standard of care for their intended use. Biomarkers could complement image-based screening in two different ways. First, they may allow refinement of screening selection criteria, independent of age and smoking habits, reducing the numbers of individuals exposed to screening and follow-up interventions. Worldwide collaborative efforts have been implemented to gain insights into the association between SNPs and common cancers (4). Lung cancer-associated single nucleotide polymorphisms (SNPs) identified in these studies may be integrated in risk models to limit the costs of lung cancer screening. Secondly, the combination of radiological findings with molecular biomarkers may facilitate the management of indeterminate pulmonary nodules (IPNs). The implementation of LDCT screening programs is rapidly increasing the detection of IPNs, which too often leads to unnecessary follow-up CTs, or even invasive procedures. Molecular markers may help to differentiate patients with malignant IPNs from the larger number of subjects with benign nodules.

      At present, no molecular biomarker of lung cancer is being used in routine clinical practice. The tremendous research efforts regarding the development and use of molecular biomarkers in lung cancer screening have recently been reviewed (5). Biomarkers can derive from cancer cells, the tumor microenvironment, or the immune response to cancer. They can be sampled from many different bodily sources, including whole blood, serum, plasma, airway epithelium, sputum, exhaled breath, or urine. Promising molecular candidates include proteins (e.g. cancer-associated antigens, autoantibodies or other immune-related markers), metabolites, microRNAs, epigenetic markers, DNA mutations or RNA signatures. Markers of this kind are at different phases of development, ranging from their analytical validation to the evaluation of their performance in the intended use population. Ultimately, evaluation of the biomarker in real clinical settings will determine its improvement of current standards and cost. Few biomarkers have reached the clinical testing phase. The application of an RNA-based signature in bronchial epithelial cell samples from the AEGIS-1 and AEGIS-2 prospective multicenter observational trials (NCT01309087 and NCT00746759) improved the diagnostic performance of bronchoscopy for the detection of lung cancer (6). In PANOPTIC (NCT01752114), a prospective multicenter observational study, plasma levels of two proteins, LG3BP and C163A, were used to discriminate benign from malignant nodules (7). The bioMILD study (NCT02247453) is prospectively evaluating the efficacy of a plasma microRNA profiling as a first line-screening test for lung cancer detection. The clinical utility of another microRNA-based signature is been validated in blood samples prospectively collected in the COSMOS-II lung cancer screening trial (8). ECLS (NCT01700257) is a randomized study aimed to assess the clinical and cost effectiveness of a test that measures a panel of seven tumor-associated autoantibodies in blood (9). The study has reached its 12,000-participant target, and initial results are expected soon. The DECAMP consortium is conducting two multicenter prospective observational trials (NCT01785342 and NCT02504697) designed to develop an integrated panel of airway and blood-based molecular markers. DECAMP-1 seeks to improve the discrimination between benign and malignant IPNs, whereas DECAMP-2 will test biomarkers to predict the development of lung cancer in screened asymptomatic high-risk individuals. Novel approaches to overcome sensitivity/specificity limitations are also being tested. Host responses to cancer based on activation of the immune system have proved to provide promising diagnostic and prognostic markers applicable in the context of lung cancer screening (10). A diagnostic signature based on the combined determination of complement-activation fragments and cancer-associated proteins has shown a notable capacity to discriminate those patients with malignant IPNs. Next-generation sequencing technologies are also starting to be applied. The CCGA study (NCT02889978) has recently concluded the enrollment of 15,000 participants (more than 10,000 of them with a diagnosis of cancer) from whom longitudinal plasma samples are being collected and analyzed by DNA sequencing and methylation profiling. This study will provide valuable information about the potential application of deep sequencing technologies in circulating cell-free DNA for the early detection of lung cancer. Finally, deep learning approaches will allow the integration of several levels of information (e.g. radiographic features, clinical characteristics and molecular biomarkers) for the generation of more accurate predictive models.

      In conclusion, molecular biomarkers are potentially useful adjuncts to LDCT screening for lung cancer, either by refining risk prior to LDCT or by assessing malignancy. A remarkable amount of discovery and clinical validation work is ongoing. However, more evidence is still needed to support the implementation of any of the proposed biomarkers in the routine clinical practice. Further development of emerging biomarkers, new technological and integrated approaches, better metrics of clinical utility, and innovative trial designs will be required to speed up the development of lung cancer early detection biomarkers.

      References:

      1. Aberle DR et al. N Engl J Med 2011; 365: 395-409.

      2. De Koning HJ et al. IASLC 19th WCLC 2018; Abstract PL02.05.

      3. Pastorino U et al. Ann Oncol 2019 [Epub ahead of print].

      4. McKay et al. Nat Genet 2017; 49: 1126-1132.

      5. Seijo et al. J Thorac Oncol 2019; 14: 343-357.

      6. Silvestri GA et al. N Engl J Med 2015; 373: 243-251.

      7. Silvestri GA et al. Chest 2018; 154: 491-500.

      8. Marzi M et al. Clin Chem 2016; 62: 743-754.

      9. Sullivan FM et al. BMC Cancer 2017; 17: 187.

      10. Ajona D et al. J Natl Cancer Inst 2013; 105: 1385-1393.

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    P1.09 - Pathology (ID 173)

    • Event: WCLC 2019
    • Type: Poster Viewing in the Exhibit Hall
    • Track: Pathology
    • Presentations: 1
    • Moderators:
    • Coordinates: 9/08/2019, 09:45 - 18:00, Exhibit Hall
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      P1.09-13 - Prognostic Value of TMPRSS4 Expression and Its Role as Diagnostic Biomarker by Liquid Biopsy in Early Stage NSCLC (ID 2499)

      09:45 - 18:00  |  Author(s): Ruben Pio

      • Abstract
      • Slides

      Background

      Relapse rates in surgically-resected non-small cell lung cancer (NSCLC) patients are between 30-45% within 5 years of diagnosis, which reflects the clinical need to identify those patients at high risk of recurrence and death. TMPRSS4 is a serine protease that plays a role in lung cancer growth, development of metastasis and resistance to chemotherapy in NSCLC models. TMPRSS4 is overexpressed through promoter hypomethylation in NSCLC tumors.

      Method

      Two cohorts of NSCLC patients (MD Anderson (MDA), n=489; and Clinica Universidad de Navarra (CUN), n=95) were used to investigate the prognostic value of TMPRSS4. The WHO 2004 classification and 8th TNM edition was used for tumor stratification. We have also developed a method to quantify he degree of TMPRSS4 and SHOX2 methylation status in liquid biopsy (plasma and bronchoalveolar lavages (BALS)) by digital droplet PCR (ddPCR), in tumor-free individuals and patients with NSCLC.

      Result

      High levels of TMPRSS4 were significantly associated with reduced relapse-free survival (RFS, p<0.001) and overall survival (OS, p<0.001) in the MDA cohort, and with OS in the CUN cohort (p<0.049). In univariate Cox regression analysis using the MDA cohort, high TMPRSS4 levels were RFS (HR=2.09; 95% IC [1.53-2.87], p<0.001) and OS (HR=1.82; 95% IC [1.38-2.41], p<0.001). In multivariate analyses, TMPRSS4 was found as an independent prognostic factor for both RFS (HR=1.82, IC [1.28-2.60], p<0.001) and OS (HR=1.44, IC [1.07-1.94], p<0.014).
      In our MDA cohort, stage IA and stage IB showed no statistical differences for RFS (p=0.27) or OS (p<0.001). However, when considering the protein expression of TMPRSS4 we were able to substratify stage IA patients in low and high risk patients, since those with high TMPRSS4 levels showed a significantly reduced RFS (p=0.002) and OS (p<0.001). Similar tendency was observed for stage IB, although statistical differences were not found.

      After successful establishment of the ddPCR conditions for TMPRSS4 and SHOX2 methylation status, we analyzed plasmas and BALS in case-control studies. In BALS (79 NSCLC patients and 26 controls), significant hypomethylation (p<0.01) was found for TMPRSS4 in the case of patients with early stage NSCLC in comparison with controls, with an AUROC of 0.72 (95% IC, 0.57-0.87) (p=0.008). SHOX2 was significantly hypermethylated in BALS from early stage NSCLC compared to controls (p<0.01), with an AUROC of 0.71 (95% IC, 0.56- 0.86) (p=0.01). In the case of plasmas (89 NSCLC patients and 25 controls): in early stages, a significant hypomethylation was found for TMPRSS4 (p<0.05), with an AUROC of 0.73 (95% IC, 0.54-0.90) (p=0.015). For SHOX2, only late stages NSCLC showed significant hypermethylation with respect to controls (p<0.05), with an AUROC of 0.68 (95% IC, 0.54-0.80) (p=0.025).

      Conclusion

      High TMPRSS4 levels are associated with worse prognosis in NSCLC patients. TMPRSS4 expression significantly discriminates patients with higher risk of disease progression and poor survival outcome in early stage NSCLC. Methylation status of TMPRSS4 can be used in both plasma and BALS to identify patients with NSCLC.

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    P2.03 - Biology (ID 162)

    • Event: WCLC 2019
    • Type: Poster Viewing in the Exhibit Hall
    • Track: Biology
    • Presentations: 1
    • Moderators:
    • Coordinates: 9/09/2019, 10:15 - 18:15, Exhibit Hall
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      P2.03-38 - Identification of a Novel Synthetic Lethal Vulnerability in Non-Small Cell Lung Cancer by Co-Targeting TMPRSS4 and DDR1 (ID 2191)

      10:15 - 18:15  |  Author(s): Ruben Pio

      • Abstract
      • Slides

      Background

      Synthetic lethality has been defined as the inability of cells to proliferate when co-targeting two genes, with a synergistically superior inhibition than that found for each individual gene. Consistent co-expression of two genes involved in a similar function is a predictor of synthetic lethality, a strategy that is being applied to find out novel cancer vulnerabilities.

      Method

      Large-scale bioinformatics analyses across 5 public databases were used to identify genes consistently co-expressed with TMPRSS4, a novel therapeutic target that we have previously identified in NSCLC. Pyrosequencing was used to evaluate methylation levels in patients and cell lines. Functional in vitro experiments and animal models were used to assess synthetic lethality of TMPRSS4 and DDR1 in NSCLC.

      Result

      Consistent co-expression between TMPRSS4 and DDR1 was found in all NSCLC databases evaluated. Similar to TMPRSS4, DDR1 promoter was hypomethylated in NSCLC in 3 independent cohorts and hypomethylation was an independent prognostic factor of disease-free survival. Treatment with 5-azacitidine increased DDR1 levels in cell lines, suggesting an epigenetic regulation. Cells lacking TMPRSS4 were highly sensitive to the cytotoxic effect of the DDR1 inhibitor dasatinib. TMPRSS4/DDR1 double knock-down cells, but not single knock-out cells suffered a G0/G1 cell cycle arrest with loss of E2F1 and cyclins A and B, increased p21 levels and apoptosis. Moreover, double knock-down cells were highly sensitized to cisplatin, which caused massive apoptosis (~40%). In vivo studies demonstrated tumor regression in mice injected with double knock-down-injected cells and lack of 18FDG-uptake by microPET analysis.

      Conclusion

      We have identified a novel vulnerability in NSCLC resulting from a synthetic lethal interaction between DDR1 and TMPRSS4. This may help designing therapeutic strategies to impair NSCLC growth by co-targeting both genes.

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