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Ignacio Gil-Bazo



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    MS 05 - Clinical Issues of Immune Checkpoint Inhibitors (ID 527)

    • Event: WCLC 2017
    • Type: Mini Symposium
    • Track: Immunology and Immunotherapy
    • Presentations: 1
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      MS 05.06 - Brain Metastasis: Rationale and Efficacy with IO (ID 7662)

      15:45 - 17:30  |  Presenting Author(s): Ignacio Gil-Bazo

      • Abstract
      • Presentation
      • Slides

      Abstract:
      The development of metastases from a primary tumor is the ultimate cause of death in most of patients with cancer. In a non-small cell lung cancer (NSCLC) series of 236 patients with advanced disease, brain metastasis appeared to be the second leading site of metastasis after bone (1). In fact, 31.8% of the patients presented brain metastasis at onset and a total of 42.8% of all patients included developed brain metastasis at any time point (1). Despite recent advances in the treatment of patients with NSCLC, CNS infiltration remains a frequent complication leading to impaired quality of life and shortened survival among NSCLC patients. In the era of personalized medicine for patients harboring oncogenic driver mutations, CNS infiltration has become an especially relevant clinical issue. In fact, an apparent higher incidence of brain metastasis at onset, among patients with molecular alterations, in particular for ALK + tumors has been reported (2). In addition, a higher cumulative incidence of CNS involvement overtime during treatment with targeted agents has been shown and CNS may be the only site of progression to first or second-line tailored therapies. Unfortunately however, most patients with advanced NSCLC present tumors lacking druggable oncogenic driver aberrations. In 2011, the tumors’ capacity of avoiding immune destruction was recognized as a new emerging hallmark of cancer. More recently, the constellation of interactions between tumor cells and the immune environment around them has definitely emerged as a potential and valuable source of innumerable novel anti-cancer targets. From the clinical perspective, treatment with immune checkpoint inhibitors targeting programmed-cell death 1 (PD-1) and its ligand (PDL-1) represent the best treatment option for many patients with advanced NSCLC in first-line (3) and most of them in second-line (4-6) due to an improved quality of life and a significant survival benefit. Unfortunately, most phase II randomized/phase III clinical trials assessing the efficacy of monoclonal antibodies against PD-1 (3,4,6) and PDL-1 (5) immune checkpoints in second or first-line settings excluded patients with active or untreated brain metastasis. Additionally, no data on the evolution of previously locally treated CNS lesions during immunotherapy have been provided, nor information revealing the sites of distant progression in patients on the study arm experiencing progression disease to the treatment. The only available results in this regard are reflected in KEYNOTE-024 trial in which the subgroup analysis showed a statistically significant benefit in terms of progression-free survival for patients without baseline brain metastasis on pembrolizumab compared to non-significant differences among patients with brain involvement receiving the PD-1 blocker (3). More interestingly, Goldberg et al. reported the only direct evidence on the potential activity of an immune checkpoint inhibitor against brain metastasis from NSCLC in a non-randomized, open label, phase II clinical trial (7). The study enrolled 36 patients with untreated brain metastases from melanoma (18 patients) or NSCLC (18 patients) receiving pembrolizumab monotherapy. Patients were given 10 mg/kg pembrolizumab every 2 weeks until progression and the primary endpoint was brain metastasis response assessed in all treated patients. In the preliminary results reported, a brain metastasis response was achieved in four (22%; 95% CI 7–48) of 18 patients with melanoma and six (33%; 14–59) of 18 patients with NSCLC. Despite the low number of patients included, this remarkable activity shown by pembrolizumab among brain metastases in patients with NSCLC warrants further investigation in larger series and prompt the analysis of the anatomic, molecular and immune factors involved in those responses. Other studies have focused their attention in the immune microenvironment of the brain in an attempt to unravel the theoretical activity of an immune-directed therapeutic approach. Berghoff at al. investigated tumor-infiltrating lymphocytes (TIL) subsets and their prognostic impact in 116 brain metastases (BM) from different tumor type’s specimens using immunohistochemistry for CD3, CD8, CD45RO, FOXP3, PD1 and PD-L1 (8). Interestingly enough, they found TIL infiltration in 115/116 (99.1%) BM specimens. PD-L1 expression was evident in 19/67 (28.4%) BM specimens and showed no correlation with TIL density (p > 0.05). High infiltration was most frequently observed for CD3+ TILs (95/116; 81.9%) and least frequently for PD1+ TILs (18/116; 15.5%; p < 0.001). Highest TIL density was observed in melanoma, followed by renal cell cancer and lung cancer BM (p < 0.001). More importantly, the density of CD3+, CD8+, and CD45RO+ TILs showed a positive and significant correlation with favorable median overall survival (OS) times. The same group has found TIL infiltration and PD-L1 expression as a common feature in Small-cell Lung Cancer (SCLC) BM. In addition, the presence of CD45RO+ memory T-cells and PD-L1+ TILs in SCLC BM seemed to be associated with favorable survival times suggesting an active immune microenvironment in SCLC BM (9). Takamori et al., evaluated the discordance in PD-L1 expression between primary and metastatic lesions and analyzed the association between the discordance and other clinical factors in 21 NSCLC patients (10). Remarkably, among the 16 patients with brain metastases, in three of them there was a good correlation in PDL-1 expression between the primary tumor and the brain metastasis. In other two patients however, positive PDL-1 primary lesions produced PDL-1 brain metastases. Interestingly enough, in two patients with PDL-1 negative NSCLC undergoing radiation therapy for brain metastases followed by surgical resection of BM, irradiated lesions turned to be positive for PDL-1 expression suggesting a potential capacity of radiotherapy to induce PDL-1 expression in BM (10). In fact, these observations along with several preclinical findings have paved the way for the design of clinical trials combining radiation therapy and immune checkpoint inhibitors against brain metastases. In summary, here we present and discuss the most relevant evidences about the particular immune microenvironment of the brain, the clinical activity of immune checkpoint inhibitors against NSCLC brain metastasis as well as their potential combination with local radiation therapy and the hypothetical use of TIL infiltrates and PDL-1 expression as predictive biomarkers for response of brain metastases to immunotherapy. Several clinical cases illustrating these evidences will be also presented and discussed. References 1. Castanon E, Rolfo C, Vinal D, Lopez I, Fusco JP, Santisteban M, et al. Impact of epidermal growth factor receptor (EGFR) activating mutations and their targeted treatment in the prognosis of stage IV non-small cell lung cancer (NSCLC) patients harboring liver metastasis. J Transl Med. 2015;13:257. 2. Kang HJ, Lim HJ, Park JS, Cho YJ, Yoon HI, Chung JH, et al. Comparison of clinical characteristics between patients with ALK-positive and EGFR-positive lung adenocarcinoma. Respir Med. 2014;108(2):388-94. 3. Reck M, Rodriguez-Abreu D, Robinson AG, Hui R, Csoszi T, Fulop A, et al. Pembrolizumab versus Chemotherapy for PD-L1-Positive Non-Small-Cell Lung Cancer. N Engl J Med. 2016;375(19):1823-33. 4. Borghaei H, Paz-Ares L, Horn L, Spigel DR, Steins M, Ready NE, et al. Nivolumab versus Docetaxel in Advanced Nonsquamous Non-Small-Cell Lung Cancer. N Engl J Med. 2015;373(17):1627-39. 5. Fehrenbacher L, Spira A, Ballinger M, Kowanetz M, Vansteenkiste J, Mazieres J, et al. Atezolizumab versus docetaxel for patients with previously treated non-small-cell lung cancer (POPLAR): a multicentre, open-label, phase 2 randomised controlled trial. Lancet. 2016;387(10030):1837-46. 6. Herbst RS, Baas P, Kim DW, Felip E, Perez-Gracia JL, Han JY, et al. Pembrolizumab versus docetaxel for previously treated, PD-L1-positive, advanced non-small-cell lung cancer (KEYNOTE-010): a randomised controlled trial. Lancet. 2016;387(10027):1540-50. 7. Goldberg SB, Gettinger SN, Mahajan A, Chiang AC, Herbst RS, Sznol M, et al. Pembrolizumab for patients with melanoma or non-small-cell lung cancer and untreated brain metastases: early analysis of a non-randomised, open-label, phase 2 trial. Lancet Oncol. 2016;17(7):976-83. 8. Berghoff AS, Fuchs E, Ricken G, Mlecnik B, Bindea G, Spanberger T, et al. Density of tumor-infiltrating lymphocytes correlates with extent of brain edema and overall survival time in patients with brain metastases. Oncoimmunology. 2016;5(1):e1057388. 9. Berghoff AS, Ricken G, Wilhelm D, Rajky O, Widhalm G, Dieckmann K, et al. Tumor infiltrating lymphocytes and PD-L1 expression in brain metastases of small cell lung cancer (SCLC). J Neurooncol. 2016;130(1):19-29. 10. Takamori S, Toyokawa G, Okamoto I, Takada K, Kozuma Y, Matsubara T, et al. Discrepancy in Programmed Cell Death-Ligand 1 Between Primary and Metastatic Non-small Cell Lung Cancer. Anticancer Res. 2017;37(8):4223-8.

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    P1.02 - Biology/Pathology (ID 614)

    • Event: WCLC 2017
    • Type: Poster Session with Presenters Present
    • Track: Biology/Pathology
    • Presentations: 1
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      P1.02-039 - Preventive and Therapeutic Action of Id1 Inhibition in KRAS-Mutant (KM) Lung Adenocarcinoma (LAC) Tumors in a Xenograft Murine Model (ID 9574)

      09:30 - 16:00  |  Author(s): Ignacio Gil-Bazo

      • Abstract
      • Slides

      Background:
      Id1 has been shown to be involved in cell viability and migration of lung cancer cell lines and confer poor prognosis in LAC-patients. The most frequently mutation in LAC is KRAS, but no targeted therapy has been successfully developed. Here we study the role of Id1 in a KM-LAC murine model.

      Method:
      The expression of Id1 was analyzed in a panel of human LAC cell lines by qPCR and Western-Blot. Several human cell lines with known mutations (H1792-604, H2009, H358, H1568, H1437, H1703, H2126) were selected to deplete Id1 expression by inducible short hairpin RNA (shRNA) regulated by doxycycline. Proliferation, cell cycle and apoptosis assays were performed to study the cellular mechanism underlying the effect of Id1 deficiency. Mouse xenograft models were generated by subcutaneous injection of KM-LAC cells (H1792-604 and H2009), both shId1 and shGFP cells, in flanks of immunodeficient mice treated with doxycycline (drinking water) from the time of inoculation or once the tumors were established.

      Result:
      Id1 overexpression was observed in 11 out of 12 cell lines as occurs in previously reported clinical data. Id1 inhibition was achieved in all cell lines compared to controls. In absence of Id1, proliferation assays showed a significant impairment of cell growth in KM-LAC cell lines [H1792-604 31.61% ± 3.96 (P < 0.001); H2009 52.73% ±4.74 (P < 0.001); H358 70.85% ± 8.01 (P < 0.001)]. In KM cells, a significant arrest in G2/M phase of cell cycle was observed when Id1 was inhibited whereas no significant changes were observed in wild type(WT) KRAS cells [KM 1.86 ± 0.28;WT 1.02 ± 0.05 (P < 0.001)]. KM-cells showed a significant apoptosis increase compared to WT-cells [KM-cells 1.66 ± 0.41;WT-cells 0.99 ± 0.13 (P = 0.001)]. In vivo, we observed a significant decrease in tumor volume in mice injected with H1792-604-shId1 cells (60% ± 32.39) compared to shGFP group (356.29% ± 115.32)(P < 0.001). Moreover, mice injected with H2009-shId1 cells did not develop tumors compared to control mice (168.35 ± 68.71)(P < 0.001). Activation of shId1 in established tumors induced a significant reduction of tumor volume in both xenograft models. The inhibition led to regression of 4 out of 10 tumors H1792-604 and all tumors in H2009 inoculated mice.

      Conclusion:
      These findings support a crucial role of Id1 in tumor development in KRAS-driven adenocarcinoma of the lung. Id1 targeting was proven effective in both, tumor prevention and treatment in our humanized murine model of KM LAC.

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    P1.07 - Immunology and Immunotherapy (ID 693)

    • Event: WCLC 2017
    • Type: Poster Session with Presenters Present
    • Track: Immunology and Immunotherapy
    • Presentations: 1
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      P1.07-035 - Lymphocytes and Neutrophils Count After Two Cycles and TTF1 Expression as Early Outcome Predictors During Immunotherapy (ID 10308)

      09:30 - 16:00  |  Author(s): Ignacio Gil-Bazo

      • Abstract
      • Slides

      Background:
      Non-small cell lung cancer (NSCLC) therapeutic paradigm has dramatically changed with immune checkpoint blockers. The unconventional response patterns seen in patients treated with immunotherapy (IT) make it difficult to differentiate patients who respond from non-responders early on in treatment; and biomarkers predicting clinical benefit are still lacking. As previously shown in melanoma, changes in absolute lymphocytes and neutrophils count (ALC and ANC) during IT (PD-1/PD-L1 inhibitors) may be related to response in NSCLC (Nakamuta et al, Oncotarget 2016). TTF1 expression has been associated with PD-L1 expression (Vieira et al, Lung Cancer 2016). We aimed to investigate TTF1 expression and changes in ALC and ANC after 2 cycles and their potential association with clinical outcomes to IT.

      Method:
      We enrolled 32 consecutive patients with advanced NSCLC treated with IT at Clínica Universidad de Navarra (Spain) since 2015. Radiological response was evaluated according to RECIST v1.1. The potential correlation between ALC and ANC changes during the first two cycles and response to treatment [disease control rate (DCR) vs progression] was evaluated using Student’s T-test. Fisher’s exact test was used to study the association between changes in ALC (<1,000 vs >1,000) and ANC (<4,000 vs >4,000) after 2 cycles and response to IT. TTF1 expression was correlated with IT response. Overall survival (OS) was assessed with Kaplan-Meier analysis and Log-rank test according to ALC and ANC.

      Result:
      TTF1 tumor expression in adenocarcinoma histology (n= 18) was significantly associated with response to IT (88% vs 45%, p= 0.03). Patients with ANC <4,000 after two cycles showed a longer median OS (NR vs 4.9 months; p=0.02). An ALC increase after 2 cycles was associated with DCR compared to progression (147 vs -155; p=0.05). ALC >1,000 after 2 cycles seemed to be more frequent among patients with TTF1+ tumors (82% vs 45%; p= 0.05) and among those experiencing DCR compared to progression (73% vs 58%; p=0.30).

      Conclusion:
      Our results show that ALC and ANC changes during IT and TTF1 expression may act as early predictors of clinical benefit in stage IV NSCLC patients treated with PD1/PD-L1 blockers. Our results warrant further investigation in larger series.

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    P2.02 - Biology/Pathology (ID 616)

    • Event: WCLC 2017
    • Type: Poster Session with Presenters Present
    • Track: Biology/Pathology
    • Presentations: 1
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      P2.02-036 - The Expression Pattern of CD26/DPP4 in Human Lung Cancer (ID 9319)

      09:30 - 16:00  |  Presenting Author(s): Ignacio Gil-Bazo

      • Abstract

      Background:
      Lung cancer is the leading cause of death among cancers. Despite improved surgical and novel radiation improvements, the overall prognosis remains poor. CD26/dipeptidyl peptidase 4 (DPP4) is a ubiquitously expressed transmembrane exopeptidase on the cell surfaces of many different cells including malignancies of breast, colon, and mesothelioma. Phase I data in mesothelioma with a specific antibody showed tolerability in mesothelioma patients.Our group found previously that the activity of CD26/DPP4 of lung adenocarcinoma (Adeno-CA) patients is four times higher than in normal tissue and the inhibition of CD26/DPP4 decreased the growth of lung tumors in experimental models. These data prompted us to analyze the expression of CD26/DPP4 in samples from lung cancer patients to unravel the role of CD26/DPP4 as a biomarker for lung cancer and a target for inhibition to reduce lung cancer burden. burden.

      Method:
      To identify CD26/DPP4 by immunohistochemistry (IHC), we tested four antibodies from Abcam, R/D systems, and Cell signaling technology on multi-organ tissue micro array (TMA) and human lung Adeno-CA cell lines (A549, H460, Gon8, Mai9) derived from advanced stage (IV) of human Adeno-CA. We selected the antibody from Cell signaling technology against CD26/DPP4. For the analysis of CD26/DPP4 by IHC in lung cancer samples, TMAs constructed from non-small cell lung cancer patients were used. The cohort consisted of 475 patients (Adeno-CA: 223; Squamous carcinoma: 252). The intensity of the staining was scored from 0 to 3 in a blinded manner. To quantify CD26/DPP4 in the supernatant of human lung Adeno-CA cell lines in vitro, ELISA was performed.

      Result:
      IHC scores revealed that Adeno-CA expresses significantly more CD26/DPP4 compared to squamous carcinoma (p<0.0001). Consistent with our previous findings, early stage cancer (IA) scores significantly higher than other stages IIB (p=0.0012), IIIA (p=0.0019), and IV (p=0.02) among Adeno-CA samples. We could not find CD26/DPP4 expression on human Adeno-CA cell lines by IHC, but the secretion of the protein in supernatant stays high (A549: 20pg/ml; H460: 161pg/ml; Gon8: 74pg/ml; Mai9: 648pg/ml).

      Conclusion:
      CD26/DPP4 expression was significantly higher at early stages of Adeno-CA samples when compared to advanced stages, supporting our previous findings. From the human cell line data, we suggest that advanced cancer secretes CD26/DPP4 more actively than early stage cancers. CD26/DPP4 seems to be a substantial target for inhibition of human Adeno-CA.

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    P3.01 - Advanced NSCLC (ID 621)

    • Event: WCLC 2017
    • Type: Poster Session with Presenters Present
    • Track: Advanced NSCLC
    • Presentations: 1
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      P3.01-088c - Exosomal Amphiregulin Induce Osteoclastogenesis Through Osteoclast Differentiation Mediated by EGFR Pathway (ID 9342)

      09:30 - 16:00  |  Author(s): Ignacio Gil-Bazo

      • Abstract
      • Slides

      Background:
      Bone metastasis is the most frequent complication in NSCLC resulting in osteolytic lesions. During the bone metastasis, there is an increase on the osteoclastogenesis in detriment of the bone formation processes. Epidermal growth factor receptor (EGFR) pathway is constitutively activated in NSCLC and it is known that EGFR binds Amphiregulin (AREG) that is overexpressed in several cancers such as colon, breast and lung. Moreover, its levels in plasma derived from NSCLC patients correlate with poor prognosis. AREG was recently described as a signaling molecule in exosomes derived from cancer cell lines. Exosomes have a key role in the cell-cell communication and they were indicated as important actors in metastatic niche preparation. For this reason, in the present work, we hypothesize a role of AREG carried by exosomes derived from NSCLC cells and plasma of NSCLC patients, in osteoclast differentiation.

      Method:
      Exosomes were isolated from CRL-2868 cells, by ultracentrifugation and characterized by Western Blotting (WB) and electron microscopy analysis. AREG expression and EGFR phosphorylation was evaluated by WB in, CRL-2868 cells, exosomes and exosomes isolated from plasma derived from NSCLC patients. The osteoclasts morphology was assessed by confocal microscopy and RANKL, MMP9 and TRAP mRNA expression were measured by Real time PCR and RANKL and MMP9 secretion was evaluated by ELISA. The human biological material used in this publication was provided by [email protected] (Antwerp, Belgium; ID: BE71030031000) and Banco de muestras biologicas Centro de investigacion Medica Aplicada (CIMA) Universidad de Navarra.

      Result:
      Exosomes derived from NSCLC plasmacontains AREG that induces EGFR pathway activation in pre-osteoclasts increasing the expression of RANKL which is able to induce the expression of proteolytic enzymes, MMP9 and TRAP, well-known markers of osteoclastogenesis. AREG function has been confirmed by loss and gain experiments with recombinant and neutralazing AREG, furthermore, knockdown-AREG exosomes do not induce osteoclast differentiation. To conclude, exosomes released in plasma of NCSLC patients, contain AREG, and induce osteoclasts differentiation of human primary osteoclasts.

      Conclusion:
      Exosomal AREG induces EGFR pathway activation that can induce RANKL expression that in turn, increases the expression of MMP9 and TRAP initiating an osteolytic bone metastasis.

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